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Full text of "Pesticides In The Modern World: Risks And Benefits"

PESTICIDES IN THE 

MODERN WORLD - 

RISKS AND BENEFITS 



Edited by Margarita Stoytcheva 



INTECHWEB.ORG 



Pesticides in the Modern World - Risks and Benefits 

Edited by Margarita Stoytcheva 



Published by InTech 

Janeza Trdine 9, 51000 Rijeka, Croatia 

Copyright © 2011 InTech 

All chapters are Open Access articles distributed under the Creative Commons 
Non Commercial Share Alike Attribution 3.0 license, which permits to copy, 
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Statements and opinions expressed in the chapters are these of the individual contributors 
and not necessarily those of the editors or publisher. No responsibility is accepted 
for the accuracy of information contained in the published articles. The publisher 
assumes no responsibility for any damage or injury to persons or property arising out 
of the use of any materials, instructions, methods or ideas contained in the book. 

Publishing Process Manager Sandra Bakic 
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Image Copyright Suzanne Tucker, 2010. Used under license from Shutterstock.com 

First published September, 201 1 
Printed in Croatia 

A free online edition of this book is available at www.intechopen.com 
Additional hard copies can be obtained from orders@intechweb.org 



Pesticides in the Modern World - Risks and Benefits, Edited by Margarita Stoytcheva 

p. cm. 
ISBN 978-953-307-458-0 



OPEN ACCESS 
PUBLISHER 



INTECH 
INTECHopen 

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Contents 



Preface IX 

Part 1 Pesticides and Food 1 

Chapter 1 Role of Pesticides in Human Life 
in the Modern Age: A Review 3 

Seyed Soheil Saeedi Saravi and Mohammad Shokrzadeh 

Chapter 2 Quality of Vegetables 

and Pests Control in African Urban Cities 13 

Dembele Ardjouma, Oumarou Badini and A. Abba Toure 

Chapter 3 Differential Efficacy of Insecticides According 

to Crop Growth: The Citrus Psyllid on Citrus Plants 33 

Katsuya Ichinose, Katsuhiko Miyaji, Kunihiko Matsuhira, 
Keiji Yasuda, Yasutsune Sadoyama, Do Hong Tuan, 
Nguyen Van Hoa and Doan Van Bang 

Chapter 4 Use of Pesticides in the Cocoa Industry and 

Their Impact on the Environment and the Food Chain 51 

George Afrane and Augustine Ntiamoah 

Chapter 5 Industrial Contaminants and Pesticides in Food Products 69 

Ruud Peters, Henry Beeltje and Marc Houtzager 

Chapter 6 Pesticide Residues in Bee Products 89 

Emmanouel Karazafiris, Chrysoula Tananaki, Andreas Thrasyvoulou 
and Urania Menkissoglu-Spiroudi 

Part 2 Environmental Impacts of Pesticides 127 

Chapter 7 Ecological Effects of Pesticides 129 

James Tano Zacharia 

Chapter 8 Ecological Impacts of Pesticides 
in Agricultural Ecosystem 143 

Khalil Talebi, Vahid Hosseininaveh and Mohammad Ghadamyari 



VI Contents 



Chapter 9 Environmental Impact and Remediation 

of Residual Lead and Arsenic Pesticides in Soil 169 

Eton Codling 

Chapter 1 Arsenic - Pesticides with an Ambivalent Character 181 

Arna Shab and Catharina CroBmann 

Chapter 1 1 Freshwater Decapods and Pesticides: 

An Unavoidable Relation in the Modern World 197 

Leandro Negro, Eloisa Senkman, 
Marcela Montagna and Pablo Collins 

Chapter 1 2 Effects of Pesticides on Marine Bivalves: 

What Do We Know and What Do We Need to Know? 227 

T. Renault 

Chapter 13 Immunotoxicological Effects of Environmental 

Contaminants in Teleost Fish Reared for Aquaculture 241 

Alberto Cuesta, Jose Meseguer and M. Angeles Esteban 

Chapter 14 Using Zooplankton, Moina Micrura Kurz 

to Evaluate the Ecotoxicology of Pesticides 
Used in Paddy Fields of Thailand 267 

Chuleemas Boonthai Iwai, Atcharaporn Somparn and Barry Noller 

Chapter 1 5 Application of Some Herbal Extracts and Calcium as an 

Antidote to Counteract the Toxic Effects of Cypermethrin 
and Carbofuran in Indian Major Carp, Labeo Rohita 281 

Subhendu Adhikari, Amita Chattopadhyayand Biplab Sarkar 

Chapter 1 6 Semi Aquatic Top-Predators as Sentinels of Diversity 
and Dynamics of Pesticides in Aquatic Food Webs: 
The Case of Eurasian Otter {Lutra lutra) and Osprey 
(Pandion haliaetus) in Loire River Catchment, France 289 

Charles Lemarchand, Rene Rosoux and Philippe Berny 

Chapter 17 Is Pesticide Use Sustainable 

in Lowland Rice Intensification in West Africa? 311 

F. E. Nwilene, A. Togola, 0. E. Oyetunji, A. Onasanya, G. Akinwale, 
E. Ogah, E. Abo, M. Ukwungwu, A. Youdeowei and N. Woin 

Chapter 18 Transgenic Pesticidal Crops and the Environment: 
The Case of Bt Maize and Natural Enemies 321 

Dennis Ndolo Obonyo and John B. Ochanda Ogola 

Chapter 1 9 Colony Elimination of Subterranean Termites 

by Bait Application Using Benzoylphenylurea Compounds, 
with Special Reference to Bistrifluron 347 

Shunichi Kubota 



Contents 



VII 



Chapter 20 Camouflage of Seeds, a Control Method 
of the Bird Mortality in Grain Crops 363 

Alexandre de Almeida, Hilton Thadeu Zarate do Couto 
and Alvaro Fernando de Almeida 

Part 3 Pesticides Mobility, Transport and Fate 391 

Chapter 21 Geochemical Indicators 

of Organo-Chloro Pesticides in Lake Sediments 393 

Stephen Kudom Donyinah 

Chapter 22 Pesticides and Their Movement Surface Water 
and Ground Water 411 

Feza Geyikci 

Chapter 23 Should We Be Concerned with Long-Term Health Problems 
Associated with Pesticides in Namibian Groundwater? 423 

Benjamin Mapaniand Rainer Ellmies 

Chapter 24 Transport of Carbon Tetrachloride 

in a Karst Aquifer in a Northern City, China 435 

Baoping Han, Xueqiang Zhu, Zongping Pei and Xikun Liu 

Chapter 25 Study of the Presence of Pesticides 
in Treated Urban Wastewaters 453 

Maria P. Ormad, Natividad Miguel, Rosa Mosteo, 
Jorge Rodriguez and Jose L. Ovelleiro 

Chapter 26 Interactions Between Ionic Pesticides 

and Model Systems for Soil Fractions 471 

Florencio Arce, Ana C. Iglesias, Rocfo Lopez, 
Dora Gondar, Juan Antelo and Sarah Fiol 

Chapter 27 Behavior and Fate of Imidacloprid in Croatian 

Olive Orchard Soils Under Laboratory Conditions 489 

Dalibor Broznic, Jelena Marinic and Cedomila Milin 



Chapter 28 Effects of Low-Molecular-Weight-Organic-Acids 
on the Release Kinetic of Organochlorine 
Pesticides from Red Soil 519 

Zhao Zhenhua, Xia Liling, Wang Fang and Jiang Xin 

Chapter 29 Fate of Pesticides in Soils: 

Toward an Integrated Approach of Influential Factors 535 

Veronique Chaplain, Laure Mamy, Laure Vieuble-Gonod, 
Christian Mougin, Pierre Benoit, Enrique Barriuso and Sylvie Nelieu 



Preface 



Volume 3 of the book series "Pesticides in the Modern World" is a compilation of 29 
chapters focused on: pesticides and food production, environmental effects of 
pesticides, and pesticides mobility, transport and fate. 

The first book section (Chapters 1-6) addresses the benefits of the pest control for crop 
protection and food supply increasing, but also the associated risks of food 
contamination. The advantages and the disadvantages of pesticides using in modern 
agriculture, and the effectiveness of their alternatives are comprehensively reviewed in 
Chapter 1. The objective of Chapter 2 is to assess the impacts of agrochemicals 
application on plants' pests and of some essential production factors on the quality of 
the vegetables produced in African urban settings. Chapter 3 reports research results 
on the efficacy of the insecticides imidacloprid, thiamethoxam, clothianidin, 
methidathion and fenobucarb and of their mixtures against the citrus psyllid on citrus 
plants in Vietnam. Chapter 4 examines the use of pesticides in cocoa production in 
Ghana, demonstrates that the current unsustainable agricultural practices create 
environmental and economic risks, and identifies improvement options. Chapter 5 
provides data on industrial chemicals and organochlorine pesticides contamination of 
food. Chapter 6 documents the contamination of bee products with pesticides and 
presents information on the sources of contamination. 

The second book section (Chapters 7-20) is dedicated to the environmental pesticides 
impacts. Chapter 7 comments on a number of pesticides ecological effects such as: 
effects involving pollinators, effects on nutrient cycling in ecosystems, effects on soil 
erosion, structure and fertility, and effects on water quality. The impacts of the 
pesticides in agricultural ecosystems, in terms of pesticides resistance development are 
discussed in Chapter 8. The occurrence of the inorganic pesticide lead arsenate in the 
environment, the pathways of its uptake, the methods for lead and arsenic toxicity 
assessment, and the contaminated soil remediation constitute the subject of Chapter 9. 
The effects of arsenic exposure are commented in details in Chapter 10. 

Chapters 11-13 covers the genotoxic and immunotoxic effects of pesticides on the 
aquatic fauna: decapods, bivalves, and teleost fish. Using zooplancton to evaluate the 
ecotoxicity of the main pesticide applied in paddy field is discussed in Chapter 14. 
Investigations on the capacity of some herbal extracts and calcium to counteract the 



X Preface 



pesticidal effects on Indian Major Carp are reported in Chapter 15. Comments 
concerning the assessment of the pesticides residues in otters and ospreys, considered 
as good sentinels and indicator species of contamination, bioaccumulation and 
biomagnification of toxic contaminants in rivers, estuaries, reservoirs and lakes are 
provided in Chapter 16. 

Chapter 17 presents cases of insecticides miss-use in rice production in West Africa 
and the related effects on the non-target organisms and the environment. The possible 
impacts of Bt maize on the development and behaviour of stem borers and their 
natural enemies are analysed in Chapter 18. Experimental data on the termiticidal 
activity of bistrifluron are reported in Chapter 19. Camouflaging of seeds treated with 
pesticides to mitigate the mortality of birds in grain crops is discussed in Chapter 20. 

The third book section (Chapters 21-29) furnishes numerous data contributing to the 
better understanding of the pesticides mobility, transport and fate. In Chapter 21 are 
presented investigations on the mode of deposition and transformation of the 
organochlorine pesticides into the sediment of Lake Liangzi in Central China. 
Chapters 22-24 address the complex phenomenon of environmental pollutants transport 
in surface and ground waters. Studies on the presence of pesticides in treated urban 
wastewaters are reported in Chapter 25. The fate of the pesticides in soils, namely the 
interaction of ionic pesticides with model systems of soil fractions, the imidacloprid 
sorption and degradation processes, and the release kinetics of organochlorine 
pesticides in the rhizosphere are discussed in Chapters 26-28. The factors involved in 
the retention and degradation of pesticides in soils are analysed in Chapter 29, 
applying an integrated approach. 

The addressed in this book issues associated with the benefits and risks of pesticides 
should attract the public concern to support rational decisions to pesticides use. The 
efforts of all the contributing authors to provide recent information are greatly 
appreciated. 



Margarita Stoytcheva 

Mexicali, Baja California 
Mexico 



Parti 
Pesticides and Food 



1 



Role of Pesticides in Human Life 
in the Modern Age: A Review 

Seyed Soheil Saeedi Saravi and Mohammad Shokrzadeh 

Department of Toxicology-Pharmacology, Faculty of Pharmacy, 
Mazandaran University ofMediccal Sciences, 

Iran 



1. Introduction 



Food production capacity is faced with an ever-growing number of challenges, including a 
world population expected to grow to nearly 10 billion by 2050 and a falling ratio of arable 
land to population. Based on evidences, in 1900 there were 1.6 billion people on the planet; 
in 1992 this had risen to 5.25 billion and by the year 2050 it will reach 10 billion. World 
population is increasing by 97 million per year. This explosive increase in world population 
is mostly in developing countries and this is where the need for food is greatest and 
starvation threatens human life; as, FAO 1 estimates that 500 million are already 
undernourished (Anon, 1990a). 

Civilization has been combating weeds, insects, diseases and other pests throughout 
history and there are many examples of how these pests have had a major impact on 
humans. One of the worst examples is the Black Plaque of Europe in the fourteenth 
century when millions died from a bacterial disease spread by fleas from rats (Hock et al., 
1991). Another example is the infamous Irish potato famine of the nineteenth century in 
which millions died and many more were forced to emigrate. A fungus also destroyed the 
entire German potato crop in the early twentieth century resulting in 700,000 deaths from 
starvation (Anon, 1992b). 

Thus, food plays a vital and strategic role in growing global population. But, food 
production is encounter to different limits. For example, there is a limit to new areas to 
cultivate; therefore we must increase agricultural production from the areas available. 
However, the specialization of production units has led to the image that agriculture is a 
modern miracle of food production (Stoytcheva, 2011). 

In our global society there is a place for people to grow and consume organic food, but if all 
our farmers decided against using farm chemicals, we would soon find ourselves in a grave 
situation. Without the use of farm chemicals, the production and quality of food would be 
severely jeopardized with estimates that food supplies would immediately fall to 30 to 40% 
due to the ravages of pests (Anon, 1990b; Anon, 1992a). While there are mountains of food 
in Europe and the US, this represents only 45 days food supply for the world. Only part of 
the problem is distribution and the ability to pay for purchases. 

^FAO 



Pesticides in the Modern World - Risks and Benefits 



While the first recorded use of chemicals to control pests back to 2500 BC, it is really only in 
the last 50 years that chemical control has been widely used (Hock et al v 1991). Many of the 
earliest pesticides were either inorganic products or derived from plants (i.e. burning 
sulphur to control insects and mites). Other early insecticides included hellebore to control 
body lice, nicotine to control aphids, and pyrethrin to control a wide variety of insects. Some 
heavy metals like lead arsenate was first used in 1892 as an orchard spray while about the 
same time it was accidentally discovered that a mixture of lime and copper sulphate 
(Bordeaux mixture) controlled downy mildew, a serious fungal disease of grapes. It is still 
one of the most widely used fungicides (Hock et al., 1991). 

Pesticides are an undeniable part of modern life, used to protect everything from flower 
gardens to agricultural crops from specific pests. Pesticides have contributed significantly to 
improving quality of life and safeguarding the environment. Although often taken for 
granted, without these important products, food production would decline, many fruits and 
vegetables would be in short supply and prices would rise. Some 20 to 40 percent of the 
world's potential crop production is already lost annually because of the effects of weeds, 
pests and diseases (according to the FAO reports) (WWW.CropLife America.mht). These 
crop losses would be doubled if existing pesticide uses were abandoned, significantly 
raising food prices. Even after harvest, crops are subject to attack by pests or diseases. Bugs, 
rodents or moulds can harm grains. In addition to increasing crop yields, crop protection 
products used in stored products can also prolong the viable life of products, prevent huge 
post-harvest losses from pests and diseases, and protect food safety for eating. 
On the other hand, although pesticides are now commonplace, concerns still exist about 
their safety and proper use. Pesticides can be used safely and effectively. But if proper care 
is not taken, pesticides can harm the environment by contaminating soil, surface and ground 
water, and ultimately kill wildlife. Also, the modern human is constantly exposed to a 
variety of toxic chemicals primarily due to changes in life style. The food we eat, the water 
we drink, the air we breathe, and the environment we live in are contaminated with toxic 
xenobiotics. Humans are exposed to such chemicals while still in the womb of the mother 
(Lederman, 1996; Rathinam et al., 2004). Therefore, human life would be threatened not only 
directly by pesticides in environment, but indirectly by contaminated food chain. 
However, the chapter tries to discuss about necessity of pesticides use in modern agriculture 
for supplying human food. Actually, traditional chemical pesticides have environmental 
inconvenience and disadvantages for human health; thus, the problems along with the 
benefits of pesticides in improvement of quality of agricultural products and food 
production and storage are mentioned. According to world's food demands and health 
hazards caused by traditional pesticides, modern and new generation of pesticides and/ or 
alternative methods to chemicals are modified to one of the most essential needs for modern 
agriculture in the present age. Some of the methods are titled in this chapter. 

2. Traditional pesticides 

2.1 What is a pesticide? 

As FAO defined, pesticide is any substance or mixture of substances intended for 
preventing, destroying, repelling or mitigating any pest, including vectors of human or 
animal disease, unwanted species of plants or animals causing harm during or otherwise 
interfering with the production, processing, storage, transport or marketing of food, 
agricultural commodities, wood and wood products or animal feedstuffs, or substances 



Role of Pesticides in Human Life in the Modern Age: A Review 



which may be administered to animals for the control of insects, arachnids or other pests in 
or on their bodies. The term includes substances intended for use as a plant growth 
regulator, defoliant, desiccant or agent for thinning fruit or preventing the premature fall of 
fruit, and substances applied to crops either before or after harvest to protect the commodity 
from deterioration during storage and transport. A pesticide may be a chemical substance, 
biological agent (such as a virus or bacterium), antimicrobial, disinfectant or device used 
against any pest. We use pesticides to cover a wide range of chemicals used to control insect 
pests, plant diseases, weeds, rats or other unwanted organisms. Currently, more than 800 
pesticide active ingredients in a wide range of commercial products are registered for use in 
agriculture to meet food supply demands (Stoytcheva, 2011; Food and Agriculture 
Organization of the United Nations, 2002). 

Pesticides can be classified by target organism, chemical structure, and physical state 
(Council on Scientific Affairs, American Medical Association, 1997). Pesticides can also be 
classed as inorganic, synthetic, or biologicals (biopesticides) which include microbial 
pesticides and biochemical pesticides. Plant-derived pesticides (botanicals), which have 
been developing quickly, include pyrethroids, rotenoids, nicotinoids, and a fourth group 
that includes strychnine and scilliroside (Kamrin, 1997). In addition, Pesticides can be 
classified based upon their biological mechanism function or application method. Basically, 
agricultural pesticides are divided into five categories, depending on the target pest (WWW. 
Humpath.com): 
i. insecticides, 
ii. herbicides, 
iii. fungicides, 
iv. rodenticides, 
v. and fumigants. 

All pesticides are toxic to some plant or rodent species; at higher doses, they can also be 
toxic to farm animals, pets, and humans. In general, prominent insecticide families include 
organochlorines, organophosphates, and carbamates. Acute toxicity of insecticides for 
mammals ranges from low to high. Herbicides used to control weeds have low acute toxicity 
for mammals; and fungicides are characterized as moderately toxic (Shokrzadeh & Saeedi 
Saravi, 2009). 

2.2 Advantages of using pesticides 

A plentiful supply of fresh products is vital for a healthy population. Numerous scientific 
studies demonstrate the health benefits of regularly eating a variety of fresh fruit and 
vegetables; and consumers are increasingly aware of these benefits. Agricultural 
productivity is a key to ensuring that this demand can be met at an affordable price; and 
crop protection products help increase productivity and usable crop yields. 
The crop protection industry's primary aim is to enable farmers to grow an abundant 
supply of food in a safe manner and prevent costs from increasing. Food production 
processes benefit from continual advancements in agricultural technologies and practices; in 
fact, a population now nearly twice as large has more food available per capita than 40 years 
ago. Tools such as herbicides, insecticides, and fungicides reduce crop losses both before 
and after harvest, and increase crop yields. 

The major benefits of pesticides and their role in food production are listed below 
(WWW.CropLife America.mht): 



Pesticides in the Modern World - Risks and Benefits 



Increase food quality and quantity: Crop protection technologies allow producers to 
increase crop yields and efficiency of food production processes. Up to 40 percent of the 
world's potential crop production is already lost annually because of the effects of 
weeds, pests and diseases. These crop losses would be doubled if existing pesticide uses 
were abandoned. In addition, pesticides allow consumers to consume high-quality 
products that are free of insect blemishes and insect contamination. Crop protection 
chemicals that reduce, eliminate, and insect damage allow the consumers to purchase 
high-quality products free of insect fragments. 

Decrease price of food: Because the use of pesticides improves crop yields, crop 
protection technologies also impact the cost of food. Without crop protection chemicals, 
food production would decline, many fruits and vegetables would be in short supply 
and prices would rise. Helping to keep food prices in check for the consumer is another 
large benefit of pesticides. 

Human health protection: Pesticides are the most effective substances to eliminate 
Insects that cause human diseases such as Malaria, Dengue fever, Lyme disease, and 
West Nile virus loom large. Also, human health is supported against insect and fungi- 
borne carcinogens, like aflatoxins, which is proceeding to hepatic and other cancers. 
Environmental protection: Other positive aspects of crop protection chemicals, in 
responsible and safe use, include household pest control, control of vegetation in 
industry and infrastructure, and recreation and protection of areas against 
environmental pests like noxious weeds, feral animals, etc, which cause land 
degradation. 

2.3 Disadvantages of using pesticides 

Food is the basic necessity of life and food contaminated with toxic pesticides is associated 
with severe effects on the human health. Hence it is pertinent to explore strategies that 
address this situation of food safety especially for the developing countries where pesticide 
contamination is widespread due to indiscriminate usage and a major part of population 
lives below poverty line. 

The four main groups of pesticides such as the organochlorine, organophosphate, 
carbamate, and pyrethroid insecticides (Smith & Gangolli, 2002; Ahmed et al., 2000) are of 
particular concern because of their toxicity and persistence in the environment; however 
several of the banned pesticides are still used on a large scale in developing countries and 
continue to pose severe health and environmental problems. Pesticide use raises a number 
of environmental concerns, and human and animal health hazards. Over 98% of sprayed 
insecticides and 95% of herbicides reach a destination other than their target species, 
including non-target species, air, water and soil (Miller, 2004). Pesticides are one of the 
causes of water pollution, and some pesticides are persistent organic pollutants and 
contribute to soil contamination. As a result, we are closely exposed to pesticides in the food 
and water we consume and in the air we breathe. Unfortunately these chemicals are non 
biodegradable, persistent and get accumulated in the environment and thus into the human 
food chain. Despite regulatory measures, these compounds continue to be detected in 
measurable amounts in the ecosystem including marine life (Smith & Gangolli, 2002). 
In addition, pesticide use reduces biodiversity, reduces nitrogen fixation (Rockets, 2007), 
contributes to pollinator decline (Hackenberg, 2007; Haefeker, 2000; Wells, 2007; Zeissloff, 
2001), destroys habitat (especially for birds) (Palmer et al., 2007), and threatens endangered 



Role of Pesticides in Human Life in the Modern Age: A Review 



species (Miller, 2004). It also happens that some of the pest adapt to the pesticide and don't 

die. What is called pesticide resistance, to eliminate the offspring of this pest, will be needed 

a new pesticide or an increase the dose of pesticide. This will cause a worsening of the 

ambient pollution problem. 

There is a growing concern that environmental chemicals, both natural and manmade, can 

cause: 

Pesticide resistance in some pests; 

Water, soil and air contamination that transfers the chemical residues along a food 

chain; 

Reduction of biodiversity and nitrogen fixation; 

Destruction of marine and birds' life and/ or genetically defects in their next 

generations; 

Changes in the natural biological balances, by means of reduction of beneficial and non- 
target organisms and insects, including predators and parasites of pests, and 

honeybees. 
On the other hand, the human population is exposed to these chemicals primarily through 
the consumption of pesticide contaminated farm products, leading to long term health 
hazards. 

Pesticides may induce oxidative stress leading to the generation of free radicals and 
alteration in antioxidant or oxygen free radical scavenging enzymes such as superoxide 
dismutase, catalase, glutothione peroxidase, glutathione reductase and glutothione 
transferase (Ahmed et al., 2000). 

Pesticide toxicity can result from ingestion, inhalation or dermal absorption. Also, many 
evidences show that pesticides are persistent in fish tissues, adipose tissue and other organs 
including brain cells, nervous system and endocrine glands, and even breast milk, etc 
(Shokrzadeh et al., 2009). Thus, continued exposure to these chemicals for a long period may 
result in various diseases listed below: 

Neurological, psychological and behavioural dysfunctions, including Symptoms of 

mild cognitive dysfunction (leading to problems in identifying words, colours or 

numbers and inability to speak fluently), Parkinson's disease (PD) (Uversky et al., 2002; 

Xavier et al., 2004); 

Hormonal imbalances, leading to infertility, breast pain, menstrual disturbances, 

adrenal gland exhaustions and early menopause (Xavier et al., 2004); 

Immune system dysfunction, leading to immune suppression that cause potentially 

serious health risks in populations highly exposed to infectious and parasitic diseases, 

and subject to malnutrition (Xavier et al., 2004); 

Reproductive system defects, including birth defects (Petrelli & Mantovani, 2002); 

Cancers, including brain cancers (i.e. neuroblastoma), soft tissue sarcomas (i.e. Ewig's 

sarcoma), and colorectal and testes carcinomas (Xavier et al., 2004); 

Genotoxicity, including DNA damage in peripheral lymphocytes (Undeger & Basaran, 

2002); 

Blood disorders, including leukaemia and non-Hodgkin's lymphoma (Zahm & Ward, 

1998; Zahm et al, 1997); 

and abnormalities in liver and kidneys, ... 
Between specific age ranges, infants and children are at great risk from the effects of 
pesticides. Several studies suggest that children may be particularly sensitive to the 



Pesticides in the Modern World - Risks and Benefits 



carcinogenic effect of pesticides. There is a potential to prevent at least some childhood 
cancer by reducing or eliminating pesticide exposure (Zahm & Ward, 1998). 

3. Modern alternatives to traditional pesticides 

Until about four decades ago, crop yields in agricultural systems depended on internal 

resources, recycling of organic matter, built-in biological control mechanisms and rainfall 

patterns. Pesticides started a revolution in agriculture and quality improvement methods. 

The state of the art in pesticides continues to evolve and progress as time passes. But, in 

these years pesticides were very toxic and left residues in the environment for a long time. 

On the other hand, loss of yields due to pests in many crops (reaching about 20-30% in most 

crops), despite the substantial increase in the use of pesticides (about 500 million kg of active 

ingredient worldwide) is a symptom of the environmental crisis affecting agriculture. 

However, farm products must obviously be free from pesticide contamination, which is 

possible primarily through organic farming. In addition, global social awareness of proper 

and minimal need based use of these chemicals, to some extent may reduce health related 

problems (Altieri, 1995). 

Therefore, many countries established adequate regulatory safeguards over the 

manufacture, sale and use of the pesticides. For this reason, complex and costly studies were 

conducted to indicate whether the material is safe to use and effective against the intended 

pest. Despite the applications, some human disasters like which occurred in Bhopal and 

China, and long term side effects on human and animal life style and environmental 

contamination amplify the approach to find and produce modern pesticides with lower 

problems and/ or to perform alternative applications to traditional pesticides. 

IPM 2 , the use of multiple approaches to control pests, is becoming widespread and has been 

used with success in countries such as Indonesia, China, Bangladesh, the U.S., Australia, 

and Mexico (Miller, 2004). IPM attempts to recognize the more widespread impacts of an 

action on an ecosystem, so that natural balances are not upset (Daly et al., 1998). New 

pesticides are being developed, including biological and botanical derivatives and 

alternatives that are thought to reduce health and environmental risks. In addition, 

applicators are being encouraged to consider alternative controls and adopt methods that 

reduce the use of chemical pesticides. 

Pesticides can be created that are targeted to a specific pest's life cycle, which can be 

environmentally friendlier. For example, potato cyst nematodes emerge from their 

protective cysts in response to a chemical excreted by potatoes; they feed on the potatoes 

and damage the crop. [81] A similar chemical can be applied to fields early, before the 

potatoes are planted, causing the nematodes to emerge early and starve in the absence of 

potatoes (WWW. Wikipedia.com). 

The major alternatives to traditional chemical pesticides are listed below: 

i. Natural pesticides, 

ii. Biological pest control, 

iii. Plant genetic engineering, 

iv. Interfering with insect breeding, 

v. Application of composted yard waste, 



2 Integrated pest management 



Role of Pesticides in Human Life in the Modern Age: A Review 



vi. Cultivation practices, 

vii. Release of organisms that fight the pests, 

viii. Interfering with insects' reproduction, 

ix. Soil steaming, etc. 

In the last 10 years, one line of research has been in the area of natural pesticides. This 

typically means that certain botanical plant oils have been processed, combined, or 

concentrated into pesticides. These plant oils have a unique action that targets a key 

neurotransmitter receptor called octopamine which is found in all invertebrates (i.e. insects), 

but not in mammals. 

Alternatives to pesticides are available and include methods of cultivation, use of biological 

pest controls (such as pheromones and microbial pesticides), plant genetic engineering, and 

methods of interfering with insect breeding (Miller, 2004). Application of composted yard 

waste has also been used as a way of controlling pests (McSorley & Gallaher, 1996). These 

methods are becoming increasingly popular and often are safer than traditional chemical 

pesticides. In addition, EPA is registering reduced-risk conventional pesticides in increasing 

numbers. 

Cultivation practices include polyculture (growing multiple types of plants), crop rotation, 

planting crops in areas where the pests that damage them do not live, timing planting 

according to when pests will be least problematic, and use of trap crops that attract pests 

away from the real crop. In the U.S., farmers have had success controlling insects by 

spraying with hot water at a cost that is about the same as pesticide spraying (Miller, 2004). 

Release of other organisms that fight the pest is another example of an alternative to 

pesticide use. These organisms can include natural predators or parasites of the pests. 

Biological pesticides based on entomopathogenic fungi, bacteria and viruses cause disease 

in the pest species can also be used (Miller, 2004). 

Interfering with insects' reproduction can be accomplished by sterilizing males of the target 

species and releasing them, so that they mate with females but do not produce offspring 

(Miller, 2004). This technique was first used on the screwworm fly in 1958 and has since 

been used with the medfly, the tsetse fly, and the gypsy moth. However, this can be a costly, 

time consuming approach that only works on some types of insects (Miller, 2004). 

Another alternative to pesticides is the thermal treatment of soil through steam. Soil 

steaming kills pest and increases soil health. 

3.1 Effectiveness of alternatives to traditional pesticides 

Some evidence shows that alternatives to pesticides can be equally effective as the use of 
chemicals. The experiences resulted from some countries used alternatives emphasize that 
reduction of pesticide use, application of composted yard waste with high carbon to 
nitrogen ratio to agricultural fields, etc were highly effective at r increasing crop yield. As a 
result, today's pesticides and alternative methods are safer and more effective in controlling 
pests than ever before in our history. 

3.2 Problems of modern pesticide systems 

As agricultural modernization progressed, the ecology-farming linkage was often broken as 
ecological principles were ignored and/ or overridden. In fact, several agricultural scientists 
have arrived at a general consensus that modern agriculture confronts an environmental 
crisis. A growing number of people have become concerned about the long-term 



10 Pesticides in the Modern World - Risks and Benefits 

sustainability of existing food production systems. Evidence has accumulated showing that 
whereas the present capital- and technology-intensive farming systems have been extremely 
productive and competitive; they also bring a variety of economic, environmental and social 
problems. Evidence indicates, however, that excessive reliance on monoculture farming and 
agro-industrial inputs, such as capital-intensive technology, pesticides, and chemical 
fertilizers, has negatively impacted the environment and rural society. Most agriculturalists 
had assumed that the agroecosystem/ natural ecosystem dichotomy need not lead to 
undesirable consequences, yet, unfortunately, a number of ecological diseases have been 
associated with the intensification of food production. They may be grouped into two 
categories: 
i. diseases of the ecotope, which include erosion, loss of soil fertility, depletion of nutrient 

reserves, salinization and alkalinization, pollution of water systems, loss of fertile 

croplands to urban development; 
ii. diseases of the biocoenosis, which include loss of crop, wild plant, and animal genetic 

resources, elimination of natural enemies, pest resurgence and genetic resistance to 

pesticides, chemical contamination, and destruction of natural control mechanisms. 

4. Conclusion 

Agricultural and veterinary chemicals are vital to our welfare and the protection of the 
health of our families and pets. Unless, and until, better, more efficient and more cost 
effective means of pest control are developed, farm chemicals will remain a major weapon 
in our constant battle against pests. Production would drop drastically, and food would be 
of poorer quality, more expensive and in short supply. Many pets and farm animals would 
suffer and die needlessly. World's economy and our standard of living would rapidly 
decline. In addition, to elevate the human life quality level and to protect public health 
against even mortal effects of chemicals, new generations of pesticides and alternatives to 
traditional chemical pesticides are applied to produce healthier and larger amount of 
various food. 

5. References 

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(Zingiber officinalis Rose) on oxidative stress induced by malathion in rats. Food and 

Chemical Toxicology 38: 443-450. 
Altieri, MA. (1995). Agroecology: the science of sustainable agriculture. Westview Press, 

Boulder. 
Anon (1990a). Evidence of the Bureau of Rural Resources to the Senate Select Committee 

inquiry into the use of agricultural and veterinary chemicals in Australia. 
Anon (1990b). Submission by the Agricultural and Veterinary Chemicals Association to the 

Senate Select Committee inquiry into the use of agricultural and veterinary 

chemicals in Australia. 
Council on Scientific Affairs, American Medical Association (1997). Educational and 

Informational Strategies to Reduce Pesticide Risks, Vol. 26, No. 2. Preventive 

Medicine. 
Daly, H., Doyen, J.T. & Purcell, A.H. (1998). Introduction to insect biology and diversity, 2nd ed. 

(Chapter 14, pp. 279-300) Oxford University Press. New York, New York. 



Role of Pesticides in Human Life in the Modern Age: A Review 1 1 

Food and Agriculture Organization of the United Nations (2002). International Code of 

Conduct on the Distribution and Use of Pesticides. 
Hackenberg, D. (2007). Letter from David Hackenberg to American growers from March 14, 

2007. Plattform Imkerinnen- Austria. URL: WWW.imkerinnen.at. 
Haefeker, W. (2000). Betrayed and sold out-German bee monitoring. URL: 

WWW.beekeeping.com. 
Hock, W., et al. (1991). Farm Chemicals Manual: A Guide to Safe Use and Handling. The 

Agricultural and Veterinary Chemicals in Association of Australia Ltd. 
Kamrin, M.A., (1997). Pesticide Profiles: toxicity, environmental impact, and fate. CRC Press. 
Lederman, S.A. (1996). Environmental contaminants in breast milk from the Central Asian 

Republics. Reproductive Toxicology 10, 93-104. 
McSorley, R. & Gallaher, R.N. (1996). Effect of Yard Waste Compost on Nematode Densities 

and Maize Yield. Journal of hematology 2(4S): 655-660. 
Miller, G.T. (2004), Sustaining the Earth, 6th ed. Thompson Learning, Inc. Pacific Grove, 

California. 
Palmer, W.E., Bromley, P.T. & Brandenburg, R.L. (2007). Wildlife & pesticides-Peanuts. North 

Carolina Cooperative Extension Service. 
Petrelli, G. & Mantovani, A. (2002). Environmental risk factors and male fertility and 

reproduction. Contraception 65:297-300. 
Rockets, R. (2007). Down On The Farm? Yields, Nutrients And Soil Quality. URL: 

WWW.Scienceagogo.com. 
Shokrzadeh, M., Saeedi Saravi, S.S. & Zehtab Yazdi, Y. (2009). Lindane residues in cultivated 

cucumber and in the most consumed fish in Caspian Sea (Iran). Toxicology and 

Industrial Health 25(8): 517-523. 
Shokrzadeh, M. & Saeedi Saravi, S.S. (2009). Fundamental Toxicology. MAZUMS Publications 

(Avay-e-Masih publication), ISBN: 978-964-2769-15-5, Iran. 
Smith, A.G. & Gangolli, S.D. (2002). Organochlorine chemicals in seafood: occurrence and 

health concerns. Food and Chemical Toxicology 40:767-779. 
Stoytcheva, M. (2011). Pesticides - Formulations, Effects, Fate. In: Pesticides in Agricultural 

Products: Analysis, Reduction, Prevention, Shokrzadeh, M. & Saeedi Saravi, S.S. (Ed), 

225-242, Intech open access publisher, ISBN 978-953-307-532-7, Rijeka, Croatia. 
Undeger, U. & Basaran, N. (2002). Assessment of DNA damage in workers occupationally 

exposed to pesticide mixtures by alkaline comet assay. Archives of Toxicology 76: 

430-6. 
Uversky, V.N., Li, J., Bower, K. & Fink, A.L. (2002). Synergestic effects of pesticides and 

metals on the fibrillation of a-synuclein: implications for Parkinson's disease. 

Neurotoxicology 23:527-536. 
Wells, M. (2007). Vanishing bees threaten U.S. crops. URL: WWW.bbc.co.uk. 
WWW. CropLife America.mht. Increasing Food Production and Crop Yields: Benefits of 

Pesticides. 
WWW. Humpath.com. Pesticides. 
WWW. Wikipedia.com. Pesticides. 
Xavier, R., Rekha, K. & Bairy, K.L. (2004). Health Perspective of Pesticide Exposure and 

Dietary Management. Malaysian Journal of Nutrition 10(1): 39-51. 
Zahm, H.S. & Ward, M.H. (1998). Pesticide and childhood cancer. Environmental Health 

Perspectives 106:893-908. 



12 Pesticides in the Modern World - Risks and Benefits 

Zahm, H.S., Ward, M.H. & Blair, A. (1997). Pesticides and Cancer. Occupational Medicine: 

State of the Art Reviews 12: 269-289. 
Zeissloff, E. (2001). Schadet imidacloprid den bienen (in German). URL: 

WWW. beekeeping, com . 



Quality of Vegetables and Pests Control in 

African Urban Cities 

Dembele Ardjouma 1 *, Oumarou Badini 2 and A. Abba Toure 3 

1 \jaboratoire Central d'Agrochimie et d'Ecotoxicologie, 

international Research & Development, Washington State University, 

3 Departement Environnement et Sante, Institut Pasteur, 

ls Cote D'ivoire 
2 USA 



1. Introduction 

Urban farming or Urban gardening (Urban Agriculture) is the practice of farming in a city 
environment. This practice of food production takes place on rooftops, in backyards, in 
community gardens and in vacant public spaces in industrial countries (JOB S Ebenezer, 
2010). In the industrialized world, urban farming largely disappeared in this century in spite 
of the recent development of the green roof movement, but in the developing world it has 
persisted and since the 1970's has shown signs of increase (Nelson., 1996). Today, in the 
developing world especially in African countries, more and more people are migrating from 
rural to urban settings adding to the increase in global population in urban cities. Such 
growing urbanization has increased the demand for quantity and quality food production 
and consumption in the cities. The contribution of urban agriculture to these cities has the 
potential to improve livelihoods and provide economic growth and stability to the 
population (Nugent, 1997; Garnett, 1996). Also, organic practices can be further promoted in 
urban agriculture by transforming nutrient rich waste from landfills into organic fertilizer 
and returning it to the land (Nancy Simovic, 1998). 

In Cote d'ivoire, migration from rural areas brings into the urban areas many persons with 
very little formal education. This may result in unemployment and under-employment of a 
sizable number of people. Urban agriculture may be a way to occupy the inner city youth, 
and new migrants. 

Urban agriculture has the potential for creating micro-enterprises that can be owned and 
operated by the community members with little initial investment capital. 
Horticulture is a vital economic sector for most African countries. Cote d'ivoire fruits and 
vegetables export to EU (European Union) countries are estimated to over 360, 000 tons. In 
2007, Burkina Faso exported more than 925, 000 tons of fresh green beans. In Mali tomatoes 
production was over 17,000 tons and okra reached 8,600 tons. Despite the economic 
potential, the horticultural sector including urban agriculture is confronted to pests' attacks 
and phytosanitary problems. It needs to comply with the pesticides regulations and the 
quality control (traceability) standards which are now required by most industrials and 
export countries. Hence, the importance of the present initiative to study the problematic of 
"The Quality of Vegetables and the Pests Control in African Urban Farming". 



14 Pesticides in the Modern World - Risks and Benefits 

The main objective of this study is to assess the impact of pest on urban farming Lettuce, 
Spinach, and Turnip production, the application of agrochemicals for plant protection, and 
the quality of irrigation water. The specific objectives are (1) to evaluate the impact of 
agrochemicals application on plants' pests, (2) to determine their economic incidence, (3) to 
monitor irrigation water quality, and (4) to control some essential production factors which 
are indicators of a good standard quality production. 

2. Materials and methods 

The study was conducted near the "M'POUTO village "located around the lagoon Ebrie 
next to the district of Riviera-Golf of the city of Abidjan, the economic capital of Cote 
d'lvoire. 




Photo 1. M' POUTO village; district of Riviera-Golf in Abidjan City. Cote d'lvoire 

The experimental zone is in full sub-equatorial climate with surrounding vegetation mostly 

composed of tall grasses and scattered bushes (Photo 1). 

The area is characterized by hydromorphic and sandy soils (DUCHAUFOUR Ph, 1997). The 

climatic conditions of the study zone is characterized by four seasonal cycles: 

A big or long rainy season from May to July and a small shorter rainy season from October 

to November followed by a long dry season from December to April and a short dry season 

from August to September. The average annual rainfall is about 2500 millimeters with a 

relative humidity of 80 to 90 % . The maximum and minimum average air temperatures are 

respectively 33°C and 21°C. 

2.1 Plants 

Subsistence crops are defined as crops that may be rich in proteins or carbohydrates grown 
by a farmer principally to feed his or her family, with little or nothing left over to sell while 
urban farming crops are considered as crops supplying luxury items intended for privileged 
people (MESSIAEN CM., 1989);. Our study concerned three urban farming vegetable crops 
namely: Lettuce, Spinach and Turnip. 



Quality of Vegetables and Pests Control in African Urban Cities 



15 



2.1.1 The Lettuce, Lactuca sativa L 

The Lettuce, Lactuca sativa L. (Asteraceae or Compositaceae) is the more consumed 
vegetable in the world. There are approximately 149 varieties worldwide (CHAUX C et al., 
1994). There are two main classes of lettuce: non-head forming lettuces such as the " celtuce 
" or " lettuce - asparagus " and the head-forming lettuces such as the " Batavia " or " curly " 
cabbage lettuce (Photo 2). 

Seeds germination is normal between 0°C and 25°C, and sunlight plays a major role in the 
growth and the development process. Lettuce has a high water demand (E.J. RYDER et al, 
1976), and grows well in different types of soils presenting a steady structure with good 
water holding capacity. In general, lettuce is a moderately heavy consumer of nutrients. 
Seedlings of lettuce are planted at 2 to 4 leaf-stage in well-prepared seedbeds (trays of earth) 
ploughed at depth and mixed to manure. The application of fertilizer (NPKS) is often 
necessary and must be incorporated in the soil before planting. The growth cycle is very 
variable (45 to 100 days) depending on the variety. Agrochemicals applications (insecticides 
and fungicides) on the lettuce cultures against the pest attacks are often done in the middle 
and end of cultural cycle. 




Photo 2. Lettuce salad: Lactuca sativa (Batavia) 



2.1.2 The spinach, Spinacia oleracea L 

The spinach, Spinacia oleracea L. ( Chenopodiaceae), is named " the prince of vegetables " 
(VERGNIAUD P. 1976). It is an annual plant generally cultivated as biennial in vegetable 
gardens (Photo 3). The plant develops initially, on a very short axis, a rosette constituted 
of fifteen (15) to twenty (20) leaves. These leaves are lengthily petiolate with full limb 
more or less blighted. Mineral fertilization (NPK) is often necessary according to expected 
yields. But the poultry's liquid manures and dejections abundantly brought are very 
largely sufficient to face exports of mineral elements. Watering must be sufficiently 
abundant to satisfy the water needs of the plant. The diseases and pest management of the 
plants must be carefully and frequently controlled (LAUMONNIER R., 1978). Also, 
weeding is very important and a thinning can be practiced in case of a very dense 
germination and seedlings. Spinach usually matures in 35 to 45 days. The plant may be 
harvested from the time there are 5-6 leaves on the plant right before the seed stalk 
develops (Photo 3). The phytosanitary protection of the plants intervenes in middle and 
end of cycle (FABIEN SEIGNOBOS et al., 2000). 



16 



Pesticides in the Modern World - Risks and Benefits 




Photo 3. Spinach: Spinacia oleracea L 



2.1.3 The turnip, Brassica rapa L. var. rapa 

The turnip, Brassica rapa L. var. rapa, (Brassicaceae) is produced in specialized market 

gardening. The plant is normally bi-annual (photo 4). In its vegetative stage it is constituted 

of a basal rosette made of about fifteen leaves with real green limb and bristling with rough 

hairs (photo 4). 

According to the varietal type, it has a tuberous root of flattened, conical or cylindrical form 

and of variable color (white (photo 4), yellow, black or two-tone) (LAURENCE S et al., 

2009). One notes about thirty varieties but the range of the varieties currently cultivated is 

rather restricted (Tokyo hybridizes Fl, Chinese turnip...). Turnips are primarily cultivated 

in full field by direct seeding on fertile well-prepared seedbeds. 

The needs in mineral elements are important and sustained fertilization (NPK) is needed 

before planting and during the growth cycle. The application of manure must be done 

before planting and preferably on the previous crop in a rotation. 

The growth cycle is 40 - 70 days dependent on climatic conditions and varieties. Turnips are 

harvested as young roots by successive thinnings. The diseases control and protection of 

turnips must be regular due to frequent pest attacks. 




Photo 4. Turnip: Brassica rapa L. (White - Turnip) 



Quality of Vegetables and Pests Control in African Urban Cities 



17 



2.2 Experimental plots 

The used method is the visual trapping by colored traps to estimate the presence, the 
quantity and the quality of the individual species (RIBA et al., 1989; FISCHER et al., 1987). 
Two types of traps were put in every plot of land: 

An air trap at the level of the foliage (see Fig 1). 

A ground trap on the surface of the ground. 

PLOT 
■* * 

10 meters 



1,5 



Air trap- 

o o 

Ground trap 



Air trap 



o o 



Ground trap 



E meters 



E :meters 



1,5 



Tre ated plot Untreate d plot (C ontrol) 

Fig. 1. Experimental Plots with air and ground traps 

All these traps contain some soapy water which captures insects. The harvest of the grips is 

made every two days with change of the trapping liquid. Insects are kept in glass jars 

containing some alcohol (70 degrees) before being sent to the laboratory. 

The ground is raised to form wide mounds or ground trays of 10 meters long by 1, 5 meters 

wide Blocks. 

Every Block is formed by two plots of land or beds of 5 meters by 1, 5 meters each (see 

Figl,). One of the plots of land is treated and the other one is untreated and constitutes the 

Control plot (blank). All in all, four Blocks and eight plots of land were realized: two Blocks 

for Lettuce, one Block for Spinach and one Block for Turnips. Every plot of land contains 

two traps. 



2.3 Agrochemicals 

2.3.1 Deltamethrin: trade name DECIS (K -OTHRINE) 

Molecular formula: C22Hi 9 Br 2 N03 (WHO., 1990a, 1990b) 
Structural formula: 




(S)- a-cyano-3-phenoxybenzyl (lR,3R)-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane 

carboxylate (IUPAC) 



18 



Pesticides in the Modern World - Risks and Benefits 



Decis 25 EC is an emulsifiable concentrate of formulation (25g/l). It is approved for a wide 
variety of insects including acarina, thysanoptera and arthropods pests of the horticultural 
plants (DEMBELE A.; 2000). We made a first application on the salad lettuce at the stage 27 
days at a concentration of 0.042 g/1 (25 L of Decis in 15 1 of water) for 400 m 2 . The stage 39 
days corresponds to the second treatment by Deltamethrin with the same concentration of 
0,042 g/1 and by Maneb with the concentration of 5 g/1 representing 93,75 grams of 
CALLIMAN 80 WP for the sprayer of 15 liters for 400 m 2 . The stage 45 days: corresponds to 
the third treatment by Maneb with the same concentration of 5 g/1. 

For turnip, at the stage 18 days we have done the first treatment by Deltamethrin with the 
concentration of 0,025 g/1 (15 milliliters of DECIS 25 EC in 15 liters of water) for 400 m 2 . The 
Stage 30 days corresponds to the second treatment by Deltamethrin with the same 
concentration of 0,025 g/1 and by Maneb with the concentration of 5 g/1 representing 93,75 
grams of CALLIMAN 80 WP for the sprayer of 15 liters. The Stage 38 days of turnips 
received the same treatment as the stage 30 days. 



2.3.2 Cypermethrin: trade name Cypercal 50 EC 
Molecular formula: C22H19CI2NO3 (WHO., 1979) 

Structural formula: „, 

\ CH 3 ! 

C=CH-d7-CaOCH 

/ 1 




RS)-I-cyano-3-phenoxybenzyl (lRS)-cis-trans-3- (2,2-dichlorovinyl)-2,2- 
dimethylcyclopropanecarboxylate (IUPAQ. 

Cypermethrin 50 EC is an emulsifiable concentrate of formulation (50 g/1). It is approved 
for a broad spectrum of harmful insects (Caterpillars, Thrips, Heliothis and white flies). 
We carried out the first application on spinaches at the stage 18 days; the amount of 
application is of 0.133 g/1 (40 ml of CYPERCAL 50 EC. in 151 of water) for 400 m 2 . At the 
stage 30 days the spinaches received an amount of treatment of 0,133 g/1 in addition to 5 g/1 
of maneb (93.75g of CALLIMAN 80 WP in 15 1 of water) for 400 m 2 . 



2.3.3 Acephate: trade name Orthen 75 SP 
Molecular formula: C 4 H 10 NO 3 PS (WHO., 1976) 

Structural formula: 



o 

CH3SPNHCOCH3 
OCH3 

0,S-dimethyl acetylphosphoramidothioate (IUPAC) 



Quality of Vegetables and Pests Control in African Urban Cities 19 

Orthen 75 SP is a water-soluble powder of formulation 75% acephate. It is a systemic 
pesticide. Methadomiphos (C 2 H 8 02NPS ) is a metabolite of acephate and it is also systemic 
(DEMBELE A.; 2000). 

Molecular formula: C 2 H 8 2 NPS 

Structural formula: 



\ 



■P NH; 

■ S 



0,S,-dimethyl phosphoramidothioate ( IUPAC ) 

These two organophosphorous pesticides are both effective against a broad range of insect 

pests (sucking, biting, and mining insects) on such vegetable and crops as cabbages, cotton, 

tobacco, sugar beet, head lettuce. It is used as a pre-harvest spray at 0.5-1.5 kg/ ha. With this 

amount, protection against the insects vermin is obtained from 7 to 21 days. 

The first application on lettuce is done at at stage 27 days with the amount of 2 g/1 ( 40 g of 

Qrthen 75 SP in 15 1 of water ) for 400m2. 

At the stage 39 days the amount of treatment of lettuce is 2 g/1 in addition to 5 g/1 of maneb 

for 400 m2. 

2.3.4 Maneb: trade name Calliman 80 WP 
Molecular formula: C 4 H 6 N2S 4 Mn (WHO-1993) 

Structural formula: H 

I 
CH ? — N — C — S, 
II 

S y Mn 

CH ? — N — C— S 
2 I II 

H S 

Manganese ethylene-l,2-bisdithiocarbamate ( IUPAC ) 

Calliman 80 WP is a wettable powder of formulation 80% of maneb (dithiocarbamate), an 
effective protective fungicide against the main foliar diseases (Anthracnose, Mildew, 
Alternaria, Rhizoctonia, cercospora, Sclerotinia and Septoria). It should be applied before 
and after seeding on all three vegetables at amount of 5 g/1. Moreover the salad lettuce 
received a treatment at the stage of 45 days. 

2.4 Plants phytopathology and pests monitoring 

After each treatment, every two days we proceed: 

To the description of the general characteristics of the plants, especially the leaves, and 
we look out for visible signs of attacks and diseased plants. 



20 



Pesticides in the Modern World - Risks and Benefits 



To the counting of the insects captured in the traps for follow-up of the dynamics of the 

recolonization following the various treatments. 
The final identification of the fungus was made after observation of the samples under a 
microscope (enlarged to a size 400 times) and according to known keys of identification 
(BOTTON.B et al, 1990; KIFFER. E et al, 1997). 

2.5 Irrigated water monitoring 

We sowed under the fume hood raw water of boring in the Petri glass, on culture medium 
sterilized. The analysis consisted of identifying thermotolerant Coliform and fecal 
Streptococci and counting of the colonies of red or pink coloring of 2 to 3 millimeters (mm) 
in diameter. The criterion of assessment is fixed to 2xl0 3 . 

3. Results and discussion 

3.1 Pests assessment on lettuces 

The trapping on the level of lettuces allowed the identification of six (6) Orders grouping 
together in total forty two (42) families of insects. They are: 10 families of Beetles, 9 families 
of Hymenopterans, 10 families of Dipterans, 7 families of Hemiptera, 3 families of 
Lepidoptera and 3 families of Orthoptera. Seventy nine per cent (79 %) of these families are 
present on the untreated plot (blank) whereas 69 % meet on the treated plot with 
Deltamethrin (Tabl 1, 2, 3) (Graphic 1). 

We counted 79% of pests in the untreated lettuce plots against 69% in the deltamethrin 
treated plots with the Hemiptera representing the most important group of devastators 
pests. The pests recolonization of the field plots was done 12 days after the first 
application of acephate against 8 days with deltamethhrin. The agrochemicals 
application makes it possible to reduce by 50% the losses of production of the Lettuces 
salad (Graphic 1). 



THE COLEOPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Buprestidae 

Carabidae 

Chrysomelidae 

Coccinellidae 

Elateridae 

Hydrophilidae 

Scarabaeidae 

Staphylinidae 

Tenebrionidae 

Cicindelidae 


P 
T 
P 
T 
P 
P 
P&T 
T 
T 
T 


+ 

+ 
+ 

+ 
+ 


+ 
+ 

+ 
+ 

+ 


+ 
+ 

+ 
+ 


+ 
+ 
+ 

+ 
+ 

+ 
+ 



( - ) = Absent (+) = Present P = Pest insects T = Non -Target Insects N = Neutrals 
Table 1. Order and Insects families' identified on lettuces 



Quality of Vegetables and Pests Control in African Urban Cities 



21 



THE HYMENOPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Pompilidae 

Ichneumonidae 

Halictidae 

Vespidae 

Formicidae 
Cynipidae 
Cephidae 

Crabronidae 

Encyrtidae 


N 
T 
T 
T 
T 
P 
P 
T 
T 


+ 
+ 
+ 
+ 
+ 


+ 


+ 
+ 
+ 


+ 


THE DIPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Agromyzidae 
Drosophilidae 
Sarcophagidae 

Muscidae 

Dolichopodidae 

Chironomidae 

Diopsidae 

Stratiomyidae 

Tephritidae 

Phoridae 


P 

N 
N 
P&N 
T 
N 
P 
N 
P 
N 


+ 
+ 
+ 
+ 
+ 
+ 


+ 
+ 


+ 
+ 

+ 
+ 
+ 
+ 
+ 
+ 
+ 


- 


THE HEMIPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Cicadellidae 
Cicadidae 
Miridae 
Piesmidae 
Coreidae 
Membracidae 
Lygaeidae 


P 
P 
P 
P 
P 
P 
P 
P 


+ 
+ 
+ 
+ 


+ 


+ 
+ 
+ 
+ 

+ 
+ 


+ 
+ 
+ 



( - ) = Absent (+) = Present P = Pest insects T = Non -Target Insects N = Neutrals 
Table 2. Order and Insects families' identified on lettuces 



22 



Pesticides in the Modern World - Risks and Benefits 





THE LEPIDOPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Pieridae 

Noctuidae 

Yponomeutidae 


P 
P 
P 


+ 
+ 


- 


+ 
+ 


- 




THE 


ORTHOPTERA ORDER 




FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Gryllidae 

Acrididae 

Gryllotalpidae 


P 
P 
P 


+ 


+ 


+ 


+ 
+ 



( - ) = Absent (+) = Present P = Pest insects T = Non -Target Insects N = Neutrals 
Table 3. Order and Insects families' identified on lettuces 



14 

12 

10 

8 

6 

4 
















-1 






rr 


1 






T 


"i- 



<? ^ 



{& 



& 






X* 



# 



& 



^ 



Orders 



JP 






# 



# 



<F 



& 



v 



■ Spray Plot (PT) 
ONa Spray Plot (PnT) 



Graphic 1. Pests Control on Lettuces 



Quality of Vegetables and Pests Control in African Urban Cities 



23 



3.2 Pests sssessment on spinaches 

On the spinach Seven (7) Orders grouping together in total Thirty seven (37) families of 

insects were identified. They are: 9 families of Beetles, 9 families of Hymenoptera, 10 

families of Diptera, 4 families of Hemiptera, 2 families of Lepidoptera, 2 families of 

Orthoptera, and 1 family of Isoptera. 

84 % of these families were present on the Untreated against 57 % on the treated plot with 

Cypermethrin. 



THE COLEOPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Carabidae 

Cicindelidae 

Scarabaeidae 

Hydrophilidae 

Staphylinidae 

Coccinellidae 

Tenebrionidae 

Chrysomelidae 

Elateridae 


T 
T 
T 
P 
T 
T 
T 
P 
P 


+ 
+ 
+ 
+ 


+ 
+ 
+ 
+ 


+ 
+ 
+ 
+ 
+ 
+ 
+ 


+ 
+ 
+ 
+ 

+ 


THE HYMENOPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Pompilidae 

Ichneumonidae 

Tenthredinidae 

Sphecidae 

Crabronidae 

Vespidae 

Formicidae 

Nyssonidae 

Bethylidae 


N 
T 
P 
T 
T 
T 
T 
T 
T 


+ 
+ 
+ 


+ 
+ 


+ 

+ 
+ 

+ 
+ 
+ 


+ 

+ 
+ 



( - ) = Absent (+) = Present P = Pest insects T = Non- Target Insects N = Neutrals 



Table 4. Order and Insects families' identified on Spinach 



24 



Pesticides in the Modern World - Risks and Benefits 



THE DIPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Tachinidae 

Agromyzidae 

Dolichopodidae 

Diopsidae 
Stratiomyidae 

Muscidae 
Sarcophagidae 
Anthomyiidae 
Calliphoridae 

Phoridae 


T 
P 
T 
P 
N 
P&N 
N 
P 
N 
N 


+ 
+ 
+ 
+ 
+ 


+ 
+ 


+ 

+ 

+ 
+ 
+ 
+ 


- 


THE HEMIPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Cicadellidae 
Piesmidae 
Cicadidae 
Tingidae 


P 
P 
P 
P 


+ 
+ 
+ 


+ 


+ 
+ 
+ 


+ 
+ 

+ 



( - ) = Absent (+) = Present P = Pest insects T = Non- Target Insects N = Neutrals 

Table 5. Order and Insects families' identified on Spinach 

We obseved 84% of pests in untreated plots against 57% in plots treated with the 
cypermethrin, with the Hemiptera representing the most important group. The 
recolonization by the pests was done 10 days after the first application of cypermethrin or 
Lambdacyhalothrin. Agrochemicals application makes it possible to reduce by 25% the 
losses of production of the spinaches (Graphic 2, photo 5). 




Photo 5. Application of pesticides doesn't respected GAP 



Quality of Vegetables and Pests Control in African Urban Cities 



25 



T 

j' 1 — iz 1— I — tItt' 1 — ~t 



Spray Pfct(PT) 
D NDSprayPlot(PnT) 



■ ^ 



J? 



J> 









# 



if 



** 



V 



J? 4 









Oidera 



Graphic 2. Pests Control on Spinaches 

3.3 Pests assessment on turnips 

On the turnips we identified six (6) Orders making a total of 34 insect's families. They are: 7 
families of Beetles, 9 families of Hymenopterans, 8 families of Dipterans, 4 families of 
Hemiptera, 4 families of Lepidoptera and 2 families of Orthoptera. 

91 % of these families were present on the untreated plots whereas 62 % were on the treated 
plot (spray plot) with Deltamethrin (Tabl 6, 7, 8,). 





THE 


COLEOPTERA ORDER 




FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 


Grounds 


Areas 


Grounds 






Traps 


Traps 


Traps 


Traps 


Coccinellidae 


T 


+ 


- 


+ 


+ 


Buprestidae 


P 


+ 


- 


- 


- 


Carabidae 


T 


- 


+ 


- 


+ 


Hydrophilidae 


P 


- 


+ 


- 


+ 


Staphylinidae 


T 


+ 


- 


+ 


- 


Cicindelidae 


T 


- 


+ 


- 


+ 


Chrysomelidae 


P 


- 


- 


+ 


+ 



( - ) = Absent (+) = Present P = Pest insects T = Non -Target Insects N = Neutrals 
Table 6. Order and Insects families' identified on the turnips 



26 



Pesticides in the Modern World - Risks and Benefits 



THE HYMENOPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 


Grounds 


Areas 


Grounds 






Traps 


Traps 


Traps 


Traps 


Pompilidae 
Ichneumonidae 


N 
T 


- 


+ 
+ 


+ 


+ 


Sphecidae 
Crabronidae 


T 
T 


+ 
+ 


- 


+ 
+ 


- 


Vespidae 
Nyssonidae 
Bethylidae 

Cephidae 
Chalcididae 


T 
T 
T 
P 
T 


+ 


- 


+ 
+ 
+ 
+ 


+ 


THE DIPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 


Grounds 


Areas 


Grounds 






Traps 


Traps 


Traps 


Traps 


Sarcophagidae 
Muscidae 


N 
P&N 


+ 
+ 


- 


+ 
+ 


+ 


Agromyzidae 

Drosophilidae 

Stratiomyidae 

Lonchaeidae 


P 

N 
N 
P 


+ 
+ 


+ 
+ 


+ 
+ 
+ 
+ 


+ 
+ 


Cecidomyiidae 
Mycetophilidae 


P 

T 


- 


- 


- 


+ 
+ 


THE HEMIPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 


Grounds 


Areas 


Grounds 






Traps 


Traps 


Traps 


Traps 


Cicadellidae 


P 


+ 


+ 


+ 


+ 


Cicadidae 


P 


+ 


- 


+ 


- 


Miridae 


P 


- 


+ 


- 


+ 


Aphididae 


P 


- 


- 


+ 


+ 



( - ) = Absent (+) = Present P = Pest insects T = Non -Target Insects N = Neutrals 



Table 7. Order and Insects families' identified on the turnips 



Quality of Vegetables and Pests Control in African Urban Cities 



27 



THE LEPIDOPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Noctuidae 

Pieridae 

Lycaenidae 

Yponomeutidae 


R 
R 
R 
R 


+ 


+ 


+ 
+ 
+ 
+ 


+ 


THE ORTHOPTERA ORDER 


FAMILIES 


RELATION 


Treated Plots 


Untreated Plots 


Areas 
Traps 


Grounds 
Traps 


Areas 
Traps 


Grounds 
Traps 


Acrididae 
Gryllotalpidae 


R 
R 


+ 


+ 


+ 


+ 
+ 



( - ) = Absent (+) = Present P = Pest insects T = Non -Target Insects N = Neutrals 

Table 8. Order and Insects families' identified on the turnips 

We observed 91% of pests in untreated plots against 62% on the plots treated with 
deltarmethrin, and the Hemiptera also set up the most important group of pests. The 
pests recolonisation was done 8 days after the first application of deltarmethrin (Graphic 3). 
The first application of agrochemicals makes it possible to reduce by 42% the losses of 
turnip production. 



12 






2 in 






(A 

* Q 1 


T I 








r t 




i r . 














S3 1 

^ 2 

1 2 


r- 1 








T 






J- 




' I 




i 


H 




O Spray plot(PT) 

D No Spray plot(PnT) 




h 


1 






J* ^ J^ ^ ^ ^ 
Orders 







Graphic 3. Pests Control on Turnips 



28 



Pesticides in the Modern World - Risks and Benefits 



Overall the application of Agrochemicals significantly reduced the number and the species 
of pests on the treated plots. The Coleoptera and the Hymenoptera contain the main species 
of the predatory and natural enemies thus auxiliary (no target insect) of plants protection, 
having a significant impact on the dynamics of the populations of pests. The preservation 
of these different auxiliaries is necessary for a sustainable management of natural resources. 
Deltamethrin has a good level of selectivity with a superior advantage for the management 
of pests and the environment over the Acephate which, has a low selectivity but a wide 
range of effectiveness against insect pest and good residual activity. The preservation of the 
auxiliaries of culture in spite of the chemical treatment is essential considering the important 
role that they play in the maintenance of agro-ecological balances. 
The Lepidoptera (larva), Orthoptera and Hemiptera represent the most important group of 
insect pests, which attack and cause the highest damage in vegetable gardening of lettuce, 
spinach and turnip. However, the considerable differences in number of captured insects 
and pests found between the treated and untreated field plots show that a targeted 
application of agrochemicals against these groups of pests is efficient. 

The majority of the groups of pests which attach and cause important damage on turnip, 
spinach and lettuce can be controlled by the application of agrochemical products applying 
good agricultural practices (GAP) compatible with the protection of the environment and 
the preservation of non-target organisms. 




Photo 6. Pesticides Plastic container on the plot (Maneb) 

However, one of the biggest problems encountered by vegetable producers is their lack of 
sufficient knowledge about how to use safely the agrochemicals. Very large numbers of 
empty pesticide containers are left lying in the fields because of the lack of collection and 
disposal facilities and constitute acute potential hazards for the environment and the fauna 
due to the left-over of toxic pesticides in the containers (Photo6). 

The producers are not sufficiently aware of the risks of pesticides accumulation in 
vegetables, and the possible health problems for consumers being exposed to these risks. 
They are also often confronted with the problems of accessibility to agricultural credits. 



Quality of Vegetables and Pests Control in African Urban Cities 



29 



3.4 Plants phytopathology and water monitoring 

We identified only one pathogenic fungus on the lettuce (9 % of production). It is 
Cladosporium sp of The Amastigomycota Divion; Group ofDeuteromycete; Hyphomycetes' 
Class and Gender of Cladosporium. This fungus is the agent responsible of Cladosporium gray 
mold, but the preventive spraying of Maneb (photo 6) gives efficient protection on the 
lettuce. 

The irrigation water is characterized by the presence of micro-organisms such as 
Thermotolerant Coliform and the fecal Streptococci. Their numbers areas respectively one 
hundred fifty (150) times and one thousand (1000) times higher than the criteria for 
international standard allowed for irrigation water quality in agricultural fields (Table 9 and 
Photo 7). 



BACTERIA 


RESULTATS 


CRITERIA 


Thermotolerant Colif orm/g 


3.105 


2.103 


Faecal Streptococci/ g 


10 6 


10 3 



Table 9. Microbiology Monitoring of Irrigated Water 







Photo 7. Irrigated water quality is doubtful 

The microbiological analysis of the irrigation water highlighted an overload of 

thermotolerant Coliforms and fecal Streptococci. These bacteria which are not normally 

pathogenic are usually used to indicate the possible presence of pathogenic microfauna 

organisms. Thus their very high number compared to the threshold recommended shows a 

low water quality (Photo 7). 

The strong presence of these indicator bacteria suggests a probable presence in the 

irrigation water of very dangerous pathogenic parasites that could develop and cause very 

important damages to the plants, farmers and the consumers. 

The contaminated vegetables can cause a certain number of diseases. Particularly, the 

contaminated salads are sources of bacterial diseases such as the typhoid and paratyphoid 



30 Pesticides in the Modern World - Risks and Benefits 

fevers (Salmonella typhi/ paratyphi) whose origin comes from the excrements of the patients 
or healthy carriers (MESSIAEN CM, 1989). Other bacteria of the Salmonella species can also 
cause collective intoxications. The periodically endemic Cholera in the tropical countries, 
maybe transmitted by soiled salads. Also the bacterial dysenteria (Shigella dysenteriae) can 
be transmitted by soiled vegetables believed contaminated by the excrements. The 
preventive protection against these diseases is often done by vaccination. But the use of 
hygienic measures like disinfections with chloramphenicol, bleach into the water or the 
potassium permanganate (KMnO/i) is of primary importance. 

4. Conclusion 

The insecticides of biological origin represent an asset but their major disadvantage in 
addition to their high costs, is their instability with storage. They quickly lose their 
effectiveness and consequently any competitiveness. But the need for both safe and natural 
food products while respecting nature and maintaining a healthy environment is a very 
important concept to be considered in Integrated Pest Management (IPM). IPM can be 
defined as a combination and the reasoned use of all the methods which makes it possible to 
control or to maintain the populations of pests to a threshold economically bearable. And if 
the consumers estimate that the products are of the first rate quality, they will not hesitate to 
pay for the full price. Finally, one can reach a great effectiveness in the improvement of 
plants protection by associating the conservation of auxiliary insects with the application of 
agrochemicals and biotechnology. Our developing countries will be able certainly to benefit 
from this progress. 

5. Acknowledgments 

I wish to thank the M'Pouto village of Riviera-Golf in Abidjan City urban farming producers 
for their collaborative contribution in this work. 

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FISCHER, L. & CHAMBON, J.P. (1987) Faunistical inventory of cereal arthropods after 

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52(2a 
FOREY P. & FITZSIMONS C. (1992), Les insectes. Libraries Grund, Paris. 125p. 
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d'identification generique. INRA, Paris-France. pl61-169. 
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J.B. Bailliere, Paris VIeme. 246p. 
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Du raifort au navet du Pardailhan. Edit Plage (La) 2009. 120 pages 
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Universitaires de France (PUF) & ACCT, Paris. 580p. 
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Differential Efficacy of Insecticides 

According to Crop Growth: 

The Citrus Psyllid on Citrus Plants 

Katsuya Ichinose 1 et al*, 

wjapan International Research Centre for Agricultural Sciences, 

^Southern Horticultural Research Institute of Vietnam, 

wjapan 
3 Vietnam 



1. Introduction 

Citrus greening is a destructive disease of citrus trees in subtropical and tropical regions. 
The pathogen of this disease in Asian countries, Candidatus Liberibacter asiaticus, is 
transmitted by the citrus psyllid, Diaphorina citri Kuwayama (Huang et all, 2004). Since no 
direct control of the pathogen has been established yet, the current management of this 
disease relies on the control of the vector, especially by insecticides (Halbert & Manjunath, 
2004; Yang et al., 2006). Many studies have been performed to examine the efficacy of 
various insecticides on the control of psyllids, revealing that neonicotinoids such as 
imidacloprid or thiamethoxam are very effective on the psyllid (Hayashikawa et al., 2006; 
Yasuda et al., 2006; Srinivasan et al., 2008; Gatineau et al., 2010; Ichinose et al, 2010a). These 
insecticides are characterised by their long-continued residue effect on the pest, up to 
several months (Yasuda et al, 2007), and their reducible effects on the psyllid population can 
continue even after the concentration of the insecticides is decreased below the lethal level 
(Boinaetal., 2009). 

Important issues in the use of insecticides are 1) how frequently an insecticide should be 
applied in year, 2) how much the insecticide should be used in each application, and 3) 
when the insecticide application should be performed in the year. For the third case, 
insecticides will be unnecessary when target pests are few due to their seasonal activities or 
other factors that affect the pest population. For example, the application of insecticides in 
winter in Florida allows the omission of insecticide uses in subsequent several months when 
psyllids are likely increased (Quresh & Stansly, 2010). On the other hand, more frequent 
insecticide application may be required for the control of the psyllid in the tropics where the 
insect can maintain its population at higher densities throughout the year. Gatineau et al. 
(2010) reported that sufficient reduction of psyllids was attained either by monthly 
application of imidacloprid or fortnightly application of fenobucarb in citrus orchards of 
southern Vietnam. The weather in this region is characterised by the tropical climate with 



* Katsuhiko Miyaji 1 , Kunihiko Matsuhira 1 , Keiji Yasuda 2 , Yasutsune Sadoyama 2 , Do Hong Tuan 3 , 
Nguyen Van Hoa 3 and Doan Van Bang 3 



34 Pesticides in the Modern World - Risks and Benefits 

two distinct seasons in the viewpoint of precipitation, dry season with low precipitation 
from November to April and wet season with frequent heavy rains from May to October. 
Serious problem in the management of citrus greening by insecticides in southern Vietnam 
is raised in the cultivation of king mandarin, which is the most predominant cultivars in 
southern Vietnam. When trees grew in the stage to yield fruits, usually one and half to two 
years after planting, the efficacy of neonicotinoids is significantly reduced or even lost 
despite either the dose or the mode of application (Ichinose et al., 2010b). The control 
efficiency on the psyllid on grown trees was not implemented by either the increase of 
insecticide dose up to 100 times or the change of the application mode, not only soil drench 
but also trunk injection, trunk painting, or leaf spray. Thus, studies were urgently needed to 
establish control methods for the psyllid on grown trees. 

The following five experiments were thus performed for the control of the psyllid on the 
grown king mandarin tree: 1) the residue effect of five insecticides, imidacloprid, 
thiamethoxam, clothianidin, methidathion and fenobucarb, were tested by the spray on the 
whole body of citrus trees cultured in a net house; 2) two application modes, soil drench and 
spray, of imidacloprid and methidathion were compared in a net house; 3) imidacloprid, 
methidathion or the mixture of these two insecticides were sprayed on three year old citrus 
trees, and their efficacy on the psyllid and aphids were counted for four weeks after the 
application. In the course of these experiments, the selling of methidathion was banned in 
Vietnam. Hence, we replaced this insecticide for dimethoate, which were tested in the 
following experiments: 4) imidacloprid, dimethoate, the mixture of these two insecticides, 
pymetrozine, or dinotefuran was applied on three years old frees and the mortality of psyllids 
were compared between these treatments; 5) imidacloprid, dimethoate or the mixture of these 
two insecticides were applied on three years old citrus trees and psyllids and other citrus pests 
were counted for four weeks after the application. More efficient uses of insecticides were 
attempted to establish not only for the control of the psyllid but also other citrus pests. 

2. Experiments on the control of psyllids by insecticides 

All experiments in this report were carried out in the province of Tien Giang, southern 
Vietnam, about 70 km south-west to Ho Chi Minh. The altitude is generally less than 10 m. 
This region lies in the tropical zone with two seasons: wet season with relatively higher 
temperatures from May to October and dry season with relatively lower temperatures from 
November of the year to April of the succeeding year. Means of annual, minimum and 
maximum temperatures are 28.0°C, 23.7°C, and 32.3°C, respectively. Annual precipitation is 
about 2500 mm. Total area for fruit crops in the Mekong Delta Region of Vietnam, more 
southern areas than Ho Chi Minh, is 282,200 ha, and 77,300 ha (27.4%) area is used for citrus. 
The most common variety of citrus crops cultivated in this region is king mandarin, which is 
sensitive to citrus greening (Koizumi et al, 1997). Mean area of farm per family is about 0.5 ha. 
Various managements for citrus greening are performed in this region, and the most efficient 
management seems to be the combination of the use of disease-free seedlings and bimonthly 
application of neonicotinoids (Ichinose & Kano, 2006). Only king mandarin, Citrus nobilis 
Loureiro, was used in our studies, which were carried out under the research project for CG 
between Japan International Research Centre for Agricultural Sciences (JIRCAS) and Southern 
Horticultural Research Institute of Vietnam (SOFRI) owing to the predominance of king 
mandarin in this region and high economic return compared to the investment for the 
cultivation. All trees were originated from seedlings produced at SOFRI under the condition of 
no invasion of CG. These seedlings are referred to as disease-free seedlings hereafter. 



Differential Efficacy of Insecticides According to Crop Growth: The Citrus Psyllid on Citrus Plants 35 

2.1 Quickness in and longevity of insecticide efficacy 

This experiment was attempted to evaluate the efficiency of one organophosphate, 
methidathion, and one carbamate, fenobucarb. These two insecticides were compared with 
three neonicotinoids, imidacloprid, thiamethoxam and clothianidin, since the efficacy of the 
latter insecticides had been examined and their efficacy was already known (Ichinose et al., 
2010a). Hence, using seedlings of king mandarin, residue effect of five insecticides was 
tested: imidacloprid (0.20 g/tree), thiamethoxam (0.17 g), clothianidin (0.19 g), methidathion 
(0.40 g) and fenobucarb (0.50 g). In the end of February 2009, 30 disease-free seedlings of 
king mandarin were purchased at the division of seedling production of SOFRI, and 18 
seedlings were randomly chosen among them. These selected seedlings were randomly 
separated into six groups with the same number, three, and each group was treated on 5 
March 2009 by no insecticide or any of the above insecticides. In the application, three 
solutions were prepared for each neonicotinoid treatment: neonicotinoid was dissolved in 
water to obtain a solution of 20 ml in the total volume so that the dose in the solution would 
be as in the above quantity. Each 20 ml solution was applied to each seedling on the soil 
surface on the pot. Methidathion and fenobucarb were diluted by water 1000 times in 
volume, thus 400 ml and 500 ml respectively, and each solution was sprayed on the whole 
body of seedlings in each treatment. The above treatments provided a completely 
randomized design with three replicates in each treatment. These seedlings were 
maintained in a net house at SOFRI. 

One leaf was collected from each tree on the day of the application before spraying, one, 
two, five, 10, 15, 30, 45, 60 and 90 days after the application. Each leaf collected on each day 
was placed in a transparent plastic cup of 500 ml just after the collection. Psyllids were 
collected one day before each leaf-collection day and left in a laboratory for one day. These 
psyllids transferred in a plastic box with plaster of paris of 1 cm thickness in the bottom for 
humidity and kept there for about one day for the starvation condition. Just after the 
preparation of the leaf, two psyllids were randomly selected from the box and released in 
each cup. The survival of each psyllid in each cup was recorded at every hour until six 
hours after the release, at 8, 12, 24, 36, 48 and 72 hr. The residue effect of the insecticides was 
determined by the mortality of psyllids in each cup. The mean of the mortality of the psyllid 
in the control cup at each observation hour on each observation day was calculated and 
used to correct the psyllid mortality in cups with insecticide-treated leaves at the same 
observation time on the same day by using the Abbott's correction formula. The detail of the 
mortality calculation is described in Ichinose et al. (2010a). 

2.1.1 Efficacy of methidathion and fenobucarb 

It is difficult to determine the effective mortality attained by insecticide for the management 
of CG. At this moment, a mortality of 80% was taken as an efficient one in this study, and 
the effective period was conveniently defined as the days during which mean mortalities 
were over this level. 

Methidathion showed quick, high lethal effect from one to 10 day within 12 hr after the 
application (Fig. 1A), but such high mortalities were not attained in 15 d after the 
application (Fig. IB). High mortalities were not seen in fenobucarb treatment throughout 
this experiment. Although mortalities were increased in 12 h after the release on 1 to 10 d 
after the application, the mean mortality was lower than 80 % (Fig. 1C). Although the 
mortality at 72 hr after the release on the 15th d was a little higher than 80 %, no high 
mortalities were seen at any time on any day after this day (Fig. ID). Both methidathion and 
fenobucarb completely lost its efficacy on the 45th d. 



36 



Pesticides in the Modern World - Risks and Benefits 



(A) 



nn 


Methidathbn ':'/' 1 

£ Id // 1 

5 10d j! \ 


/ 








-■ 










SO 


hi 















AOO 













15 30 45 60 75 

Time after psyllid release (h) 




(C) 



(D) 




15 30 45 60 

Time after psyllid release (h) 




15 30 45 60 75 

Time after psyllid release (h) 



15 30 45 60 75 

Time after psyllid release (h) 



Fig. 1. Mortality of psyllids released on leaves treated with thiamethoxam one to 10 d (A) 
and 30 to 90 d (B) after the application. Similarly the mortality of psyllids on leaves treated 
with fenobucarb is shown in C and D, respectively. All mortalities were corrected by the 
Abbott's formula with using the data of the control treatment. Bars indicate the standard 
errors of the means. 



2.1.2 Efficacy of neonicotinoids 

The mortality of psyllids released on leaves treated with imidacloprid was not high until 
24 h after the release of psyllids on the leaf until the 10th day after the application (Fig. 
2A). Although the mortality reached 100 % after 72 hr even on the 1st to 5th day, it took 
also 72 hr to reach this mortality on the 60 d and no such high mortalities were attained 
thereafter (Fig. 2B). Both thiamethoxam and clothianidin needed 10 days after the 
application to reach high mortalities^ 80 %, at 72 h after the release (Fig. 2C, E) and no 
high mortalities were attained before this time throughout the experiment (Fig. 2D, F). 
Mortalities on the 90th day were lower in both thiamethoxam and clothianidin. Hence, 
three neonicotinoids showed similar quickness of both the lethal effect and the residue 
effect, although imidacloprid seemed to affect quickly psyllids in the early periods after 
the application. 

These results indicate that the application of methidathion with a frequency of every 10 days 
would control psyllids best among the insecticides examined. If any of three neonicotinoids 
are used, similar results in the control of psyllids could be expected among them. 
Application time of every two month can be recommended. Although the probability of the 
transmission of the CG pathogen by the psyllid is not known well, methidathion would 
reduce the infection more efficiently than the neonicotinoids owing to its quickness to reach 
the 100% lethal effect. If adults eclosed from nymphs grown on infected trees would 



Differential Efficacy of Insecticides According to Crop Growth: The Citrus Psyllid on Citrus Plants 37 

transmit the pathogen more efficiently than those infested by the pathogen after eclosing 
(Inoue et al. 2009), the efficacy of the CG reduction would be similar between methidathion 
and imidacloprid. Since three neonicotinoids showed similar residue effect but imidacloprid 
seemed to be better in the quickness, imidacloprid was examined in the succeeding 
experiments. 



(A) 











Imidacloprid 

5 id 

5 Sd 
;J 10 d 


,/ 


f 




A 
















15 30 45 60 75 

Time after psyllid release (h) 



(C) 



(D) 




15 30 45 60 75 

Time after psyllid release (h) 




(F) 



15 30 45 60 75 

Time after psyllid release (h) 



15 30 45 60 

Time after psyllid release (h) 



100 


Thiamethoxam 
5 30 d 
J 60 d 




-^9* 




5 90 d 








•' 


50 
















l / 




J- 


-' — 


1 -'"'"' 





**£-* 




,,■'" 


*** * " " ~ 



15 30 45 60 

Time after psyllid release (h) 



10(1 


Clothianidin 
5 30 d 

5 60 d 
J 90 d 


-v 


'lit 


--r* 


50 




t 




















15 30 45 60 75 

Time after psyllid release (h) 



Fig. 2. Mortality of psyllids released on leaves treated with imidacloprid one 10 d (A) and 30 
to 90 d (B) after the application. Similarly mortalities of psyllids on leaves treated with 
thiamethoxam and clothianidin are shown in C-D and E-F, respectively. All mortalities were 
corrected by the Abbott's formula with using the data of the control treatment. Bars indicate 
the standard errors of the means. 



2.2 Effect of the application mode of insecticide: spray and soil drench 

In this experiment, it was attempted to test the application mode of imidacloprid and 
methidathion. In particular, focused issue was the extension of the residue effect of 



38 



Pesticides in the Modern World - Risks and Benefits 



methidathion by soil drench. Insecticides sprayed on leaves would be flown away off by 
raining, while those applied on soil surface by drenching would be less affected by rain. 
Thus, the efficacy of these insecticides applied by leaf spray or soil drench was compared. 
Qn 18 March 2008, 25 seedlings of king mandarin were purchased at SOFRI, and were 
separated into five groups in the same number. Any of the following treatment was 
randomly distributed to the groups: no insecticide, spray of 0.2 g imidacloprid in 300 ml 
water, soil drench of 0.2 g imidacloprid diluted to 20 ml by water, spray of 0.40 g 
methidathion in 400 ml water, and soil drench of 0.40 g methidathion diluted to 20 ml by 
water. On the day of the seedling purchase, seedlings were treated as in the above 
experiment, and the evaluation of the efficacy of each insecticide by each application mode 
was evaluated similarly. 

2.2.1 Comparison of application mode 

The psyllid mortality at 12 h after the release was generally low, except for imidacloprid by 
spray or drench on the 10th day after the application and methidathion by spray from first 
to fifth day (Table 1). If mortality over 80% was taken as "effective" residue effect, the best 
efficacy at 12 h was attained by methidathion by spray (Table 1). The mortality at 24 h was 
still under the effective level in the treatment of imidacloprid by spray or drench on any day 
except for the 10th day, although the days over this level in the methidathion treatment by 
spray was extended until 10th day. Methidathion by soil drench was still under the level on 
any day. The psyllid mortality at 72 h after the release was over the effective level on fifth to 
30th days in the treatment of imidacloprid by spray or drench. Methidathion by spray was 
effective at this time on the first to 15th days after the application, but this insecticide by soil 
drench was effective only on 15th day. 



Insecticide 
applied 


Application 
mode 


Time 
(h) 


Max mortality 
(%) 


Start 
(d) 


End 
(d) 


Imidacloprid 


Spray 


12 


100.0 


10 


10 


Imidacloprid 


Drench 


12 


100.0 


10 


10 


Methidathion 


Spray 


12 


100.0 


1 


5 


Methidathion 


Drench 


12 


50.0 


n/a 


n/a 


Imidacloprid 


Spray 


24 


100.0 


10 


30 


Imidacloprid 


Drench 


24 


100.0 


10 


30 


Methidathion 


Spray 


24 


100.0 


1 


10 


Methidathion 


Drench 


24 


66.7 


n/a 


n/a 


Imidacloprid 


Spray 


72 


100.0 


5 


30 


Imidacloprid 


Drench 


72 


100.0 


5 


30 


Methidathion 


Spray 


72 


100.0 


1 


15 


Methidathion 


Drench 


72 


83.3 


15 


15 



Table 1. The maximum mean of the psyllid mortality observed at 12 or 72 hours after the 
release on leaves treated with imidacloprid or methidathion applied by leaf spray or soil 
drench. The first day when the mean mortality was over 80 % and the last day when the 
mean was reduced below the level again are shown as start and end, respectively. The 
treatment in which a mortality over 80% was not attained is shown by "n/a". 



Differential Efficacy of Insecticides According to Crop Growth: The Citrus Psyllid on Citrus Plants 39 

These results indicate that imidacloprid could effectively control psyllids in 72 h from 
fifth day on after the application, but the effective level would be only on 10th day unless 
it would take 24 hours to transmit pathogen efficiently. Imidacloprid by spray did not 
show quick lethal effect even at 72 h after the release of psyllid until fifth day after the 
application. It provided similar results with this insecticide by soil drench in both 
quickness and residue effect. This suggests that either permeability or systemics of 
imidacloprid would be delayed even on leaves. Methidathion by soil drench should be 
avoided, since this application would not bring good controls in either quickness of 
efficacy or longevity. In this point, methidathion by spray could be expected for better 
management than imidacloprid owing to its quickness in the termination of psyllids after 
infestation on plants. However, the relatively shorter residue effect would need more 
frequent application of the insecticide which would not be preferred by farmers. 
Although either imidacloprid or methidathion was not effective if they were applied on 
grown trees in field (Ichinose et al., 2010b), it has been reported that imidacloprid 
effectively control psyllid populations in field (cf. Srninivasan et al., 2008; Boina et al., 
2009; Gatineau et al., 2010). Thus, the application of imidacloprid and methidathion in 
field was examined also in this study. 

2.3 Application of methidathion and imidacloprid in field 

The results of the above two experiments indicate that methidathion can quickly eliminate 
psyllids probably within 24 hr after application but the efficiency could continue shorter 
than a half month. On the other hand, neonicotinoids, especially imidacloprid, could 
maintain high efficacy for longer than one month, although it would take five to 10 days 
to reach such high efficacy and three days would be needed to attain effective control 
level. These results also provide an expectation that the mixture of these two insecticides 
would have quick and long-lasting efficacy on the psyllid. Thus, the efficacy of individual 
insecticides and the mixture of them were examined in field. Two orchards, located in Cai 
Be, about 100 km south-west of Ho Chi Minh, were selected for this study. The area size of 
each orchard was extended about 0.35 ha, in which three year old king mandarin were 
planted in 2.5 m distance both between trees and lines. The total numbers of king 
mandarin trees in these two orchards were 386 and 378. The experiment in this section 
was carried out from July 2009 to June 2010. Before the study, both orchards were divided 
into four parts, in each of which three lines lied and 16 to 18 trees were planted on each 
line. Hence, these divided parts had 54 to 56 trees. Any of the following four treatments 
was randomly distributed to a part of each orchard: no insecticide application as control; 
spray of 0.40 g methidathion dissolved in 400 ml water per tree; spray of 0.20 g 
imidacloprid dissolved in 400 ml water per tree; spray of the mixture of 0.40 g 
methidathion and 0.20 g imidacloprid in 400 ml water per tree. Based on the results in the 
second experiment, high residue effect in the plots with imidacloprid was expected to be 
maintained for one month, although residue effect in the plots with only methidathion 
would be kept high less than two weeks. Thus, the insecticide spray was carried out once 
in the early of every month during the study period. In this experiment, 15 trees were 
selected systematically: every seven trees from the first tree were examined. For the 
evaluation of insecticide efficacy, psyllid adults were counted individually, and nymphs 
were counted in a unit of colony. Colony was defined as a group of nymphs on one new 



40 



Pesticides in the Modern World - Risks and Benefits 



shoot. Besides the count of the psyllid, aphids and scale insects were also counted in a 
colony unit as in the psyllid nymph. The efficacy of each insecticide treatment was 
evaluated in the numbers of these pests, compared with the control treatment. These 
counts were performed within three days after the insecticide application and once every 
week thereafter until the end of the month. The mean numbers of psyllid adults and 
nymph colonies, aphid and scale colonies were calculated for individual selected trees 
over all samplings. These numbers were used for statistical analyses to evaluate the effects 
of treatments on these insects. 

2.3.1 Insecticide efficacy on the psyllid 



(A) 



S 2 




(B) 




Jul/09 Nov/09 Mar/10 

Sep/09 Jan/10 May/10 

Sampling month (mo/yr) 



Jul/09 Nov/09 Mar/ 10 

Sep/09 Jan/10 May/10 

Sampling time (mo/yr) 



Fig. 3. Densities of psyllid adults and nymph colonies in the plot without any insecticide for 
the control of psyllids in two old orchards, CB1 (A) or CB2 (B). Bars indicate the standard 
errors of the means. 

The means of adult and nymph colony counts on each tree in every month were 
calculated. Psyllids in the control plot without insecticide in two orchards were expected 
to be free from the influence of insecitide. Then, the densities of both adults and nymph 
colonies in these plots were compared between sampling times. Both of adults and 
nymphs increased in April to June 2010, but were generally much fewer in the other 
months (Fig. 3 A, B). 

The population of psyllid adults and nymphs were compared between treatments with 
using the data obtained after April 2010. In these analyses, the means of these counts were 
calculated for every sampling time to trace the time-dependent change of the residue effect 
of insecticides. The effects of insecticide treatment on the psyllid was evaluated by 
MANOVA, in which the means of the psyllid counts were compared among orchards and 
treatments. The numbers were significantly different between orchards {F% m = 4.229, P = 
0.017) but not between treatments (F6, 222 = 1.680, P = 0.170). The interaction of orchard and 
treatment was not significant (Fs, 222 = 1.349, P = 0.237). Although there were significant 
differences in the numbers of psyllids between treatments, apparent differences in the 
numbers were hardly found in the change of the population density of the psyllid between 
the treatments through the above sampling periods (Fig. 4A - F). Furthermore, no distinct 
decreases in the psyllid numbers were seen at the first sampling time just after each 
insecticide spray. These results means that these insecticides by spray did not effectively 
reduce psyllids in the field, although the application mode and dose both followed those 
recommended as effective in the former experiments. 



Differential Efficacy of Insecticides According to Crop Growth: The Citrus Psyllid on Citrus Plants 41 



(A) 



£-,1 ^ * * ^ *^f** * * -l 



Methidathion 

5 Orchard CB1 
5 Orchard CB2 




Jul 09 Sep 09 Nov 09 Jan 10 Mar 10 May 10 
Sampling time (m/y) 



(B) 



VVvVvvVVVVVV 



Imidacloprid 

$ Orchard CB1 
5 Orchard CB2 



<T, 



M 




fcxxxxx Wt niH'HHiiiKrm ixxxxxT 
Jul 09 Sep 09 Nov 09 Jan 10 Mar 10 May 10 
Sampling time (m/y) 



(C) 



3 Methidathion + Imidacloprid 

5 Orchard CB1 
5 Orchard CB2 






o [*nr. 




Jul 09 Sep 09 Nov 09 Jan 10 Mar 10 May 10 
Sampling time (m/y) 




Jul09 Sep09 Nov09 Jan 10 Mar 10 MaylO 
Sampling time (m/y) 



(E) 



"7" J^ \^ 4' "*■ T t y/^^X^y^ 



Imidacloprid 

J Orchard CB1 
£ Orchard CB2 




Jul09 Sep 09 Nov09 Jan 10 Mar 10 MaylO 
Sampling time (m/y) 



(F) 



Mcthida thion + Imidacloprid 
5 Orchard CB1 
^ Orchard CB2 



▼ ▼ ▼ V V W V vvv 




M09 Sep09 Nov09 Jan 10 Mar 10 MaylO 
Sampling time (m/y) 



Fig. 4. Densities of adults (A-C) and nymph colonies (D-F) in two orchards in Cai Be, southern 
Vietnam. Each orchard was divided into four plots, and one was used as control without any 
insecticide and the other three plots treated by either methidathion 0.40 g/tree, imidacloprid 
0.20 g/tree or the mixture of these two insecticides in two orchards. Arrows show the time 
when insecticides were sprayed. Bars indicate the standard errors of the means. 



2.3.2 Insecticide efficacy on the aphid and scale insect 

Aphids increased in November 2009 to March 2010 in both orchards, although a smaller 
increase was seen in July 2009 in CB2 (Fig. 5 A, B). In both orchards, monthly application of 
insecticides reduced the aphid population, but the efficacy continued for one to two weeks 
only in both orchards. In particular, the aphid population seems to have been less affected by 
imidacloprid. On the other hand, scales insects increased until november 2009, and showed 
little increase thereafter (Fig. 5C, D). In CB2, scales were fewer throughout this study than in 
CB1. Thus, the scale population was effectively suppressed just after the application of 
methidathion, but the residue effect was maintained for less than two weeks. The application 
of imidacloprid led the scale rather to increase more than the no-insecticide treatment. 



42 



Pesticides in the Modern World - Risks and Benefits 



(A) 






(B) 



Orchard CB1 

5: No insecticide 

j£ Methidathion 
5 I m id ac lop rid 
5 Methidathion + ImidLicbprid 




Jul 09 Sep 09 Nov 09 Jan 10 Mar 10 May 10 
Sampling time (m/y) 



10 




| Orchard CB2 

3; No insecticide 
1 3! Methidathion 
T jjj Imidacloprid 
I ^ Methidathion + Imidacbprk 


5 


* * » * .:' 


llll 1 1 i 11 1 





A ^Jlklk 



Jul09 Sep 09 Nov09 Jan 10 Mar 10 May 10 
Sampling time (m/y) 



(C), 



Orchard CB1 

j No 

5 Methidathion 
5 Imidacloprid 

5 Methidathion + Imidudopml 




I | I | i 1 I 



(D) 6 







Orchard CB2 

5 No insecticide 
3; Methidathion 
5 Imidacloprid 

|]j Methidathion - Imidacloprid 



111111111111 



Jul 09 Sep 09 Nov 09 Jan 10 Mar 10 May 10 
Sampling time (m/y) 



Jul09 Sep09 Nov09 Jan 10 Mar 10 MaylO 
Sampling time (m/y) 



Fig. 5. Densities of aphid (A, B) and scale insect (C, D) colonies in two two-years old 
orchards, where seedlings had been planted either in May 2007 (CB1) or in November 2007 
(CB2). Each orchard was divided into four plots, to each of which any of the four treatments 
were randomly distributed: no insecticide use for the control of psyllids, 0.40 g/tree 
methidathion by spray, 0.20 g/tree imidacloprid by spray, or 0.40 g methidathion and 0.20 g 
imidacloprid/ tree by spray. Arrows indicate the time when insecticides were sprayed. Bars 
show the standard errors of the means. 

Univariate ANOVA was used to evaluate the effect of treatment on the aphid and scale. The 
mean numbers of the aphid colony was significantly different both between orchards (Fi, 112 
= 4.958, P = 0.028) and between treatments (F 3 , 112 = 3.803, P = 0.012). The interaction of these 
two variables was significant (F3, 112 = 2.754, P = 0.046). Similarly, the number of scales were 
significantly different both between orchards (Fi, 112 = 12.983, P < 0.001) and between 
treatments (F3, 112 = 8.936, P < 0.001). The interaction of these two variables was significant 
(F 3 ,ii2 = 3.718, P = 0.014). 

These results indicate that aphids could be effectively controlled by either methidathion or 
imicloprid, but the efficacy would be continued for two weeks at longest. Scale insects 
would be controlled effectively by methidathion, but imidacloprid should be avoided for 
the control of the scale. 



2.4 Efficacy of insecticides replaced for methidathion 

Unfortunately, the sale of methidathion was discontinued in 2010 in Vietnam. According to 
local vendors, this was not due to any problem of methidathion per se, but farmers were 
likely to avoid using this insecticide due to its relatively higher prices in the market of 
Vietnam. Irrespective of the reason of the ban, it was urgently needed to find other 
insecticides that could be replaced for methidathion. Candidate insecticides should have 



Differential Efficacy of Insecticides According to Crop Growth: The Citrus Psyllid on Citrus Plants 43 

been those which could be easily purchased in Vietnam and would be expected to show 
similar efficacy as methidathion. For these conditions, dimethoate was selected, since it 
belongs to the same insecticide group with methidathion, "organophosphate". Two other 
insecticides, dinotefuran and pymetrozine, were also examined as well. For this experiment, 
12 two-year old king mandarin trees were randomly selected in an orchard, located in Cai 
Be, about 120 km south-east to Ho Chi Minh. The mean (± SEM) of circumference of these 
trees just above the grafted part was 156.2 ± 22.8 mm. This experiment was consisted of four 
treatments, no insecticide, dimethoate, dinotefuran and pymetrozine, each of which was 
randomly distributed to three trees, producing three replicates for each. The doses of 
dimethoate, dinotefuran and pymetrozine were 0.40 g/tree, 0.20 g/tree and 0.50 g/tree, 
respectively. These insecticides were dissolved in water to become 400 ml in total volume, 
and sprayed on the whole body of the tree. The spray was executed on 11 May 2010, and 
there was no precipitation for longer than one month after this day. Two leaves of each tree 
were collected on the day just before the insecticide application, one, two, five, 10, 15, 20, 30, 
45, 60, and 90 days after it. These leaves were treated as in the previous experiments for the 
test of insecticide efficacy. The mean of psyllid mortality on two leaves of each tree on each 
collection day was calculated, and the mean was used as the mortality which was achieved 
at the observation time on the day. The mean of the mortality on the control tree at a given 
time on a given day was calculated, and incorporated into the Abbott's formula to correct 
the mortality on insecticide-treated leaves at the same time on the same day. 



2.4.1 Insecticide efficacy on the psyllid 

High lethal effect of dimethoate was attained only at 72 hr after the release of psyllids only 
one day after spray, and decreased thereafter (Fig. 6). The psyllid mortality at 12 hr on this 
day was only about 60%. The results mean that dimethoate was inferior in both quickness 
and residue of the efficacy on the psyllid to methidathion. Hence, it could be expected that 
field application of dimethoate would show mortalities as high as methidathion on one day 
after the spray, and its effectiveness would be much lower thereafter with allowing the 
psyllid population to be recovered earlier. Dinotefuran showed similar effect as dimethoate 
until this day, but its residue effects was likely to be higher than dimethoate thereafter. The 
mortality of psyllids at 72 hr by dinotefuran was smaller than 80% after five days. These 
results in this experiment indicate that both quickness and residue effects of dinotefuran 



(A) 



<£ Dimethoate 
2 DInotefiiran 
21 Pymetrozine 







1 5 10 15 20 30 45 60 90 
Time after spray (d) 




5 10 15 20 30 45 60 90 
Time after spray (d) 



Fig. 6. Mortality of psyllids at 24 (A) and 72 (B) hours after released on leaves collected on 
the days after application of dimethoate, dinotefuran, or pymetrozine. The psyllid mortality 
was corrected by the Abbott's formula. Bars indicate the standard error of the mean. 



44 



Pesticides in the Modern World - Risks and Benefits 



were lower than those of imidacloprid, and thus dinotefuran should be much less effective 
on the control of psyllids in field than that of imidacloprid. The lethal effect of pymetrozine 
was always lower through this experiment, never attaining mortality over 50% . Based on the 
results, dimethoate and imidacloprid were examined in the following experiment. 

2.5 Efficacy of dimethoate in field 

The efficacy of dimethoate on the psyllid in field was examined as in the experiments of 
methidathion. The two orchards which had been used for the experiment of methidathion 
were also used for this experiment. Each of the two orchards was divided into four plots, to 
each of which any of the four treatments was randomly distributed: no insecticide, spray of 
0.80 g dimethoate per tree, spray of 0.20 g imidacloprid, and spray of 0.80 g dimethoate and 
0.20 g of imidacloprid per tree. These treatments were carried out in the early of every 
month from July 2010 to November 2010. The dilution and application mode of insecticides 
were same as in the experiment of methidathion. Psyllids, aphids, and scale insects were 
counted in a couple of day after the insecticide application, and once a week after then. This 
experiment was carried out from July 2010 to November 2010. 



2.5.1 Insecticide efficacy on the psyllid 

Adults and nymphs increased in July to September 2010, but few were present in the other 
months in both orchards (Fig. 7 A, B). Although adults were likely to be more in CB1 than in 
CB2 and nymphs were more in CB2 than in CB1 in the early period of this experiment, no 
apparent differences in these numbers were seen between the two orchards thereafter. 



(A) r 

4 
£ 3 

| 2 

= 
■a 
< 1 





(B) 



5 Orchard CB1 
7> Orchard CB2 



HiOw 



Aug 10 Sep 10 Oct 10 Nov 10 

Sampling time (m/y) 




J Orchard CB1 
7* Orchard CB2 



JullO AnglO Sep 10 Oct 10 Nov 10 

Sampling time (m/y) 



Fig. 7. Densities of adult (A) and nymph colonies (B) of the psyllid in plots without any 
insecticide for the control of psyllids in two old orchards, where seedlings had been planted 
either in May 2007 (CB1) or in November 2007 (CB2). Each orchard was divided into four 
plots, to each of which any of the four treatments were randomly distributed: no insecticide 
use for the control of psyllids, 0.40 g/tree dimethoate by spray, 0.20 g/tree imidacloprid by 
spray, or 0.40 g dimethoate and 0.20 g imidacloprid/ tree by spray. Bars indicate the 
standard errors of the means. 

Despite few occurrences of psyllids in the control plot after September, both adults and 
nymphs were counted at higher densities in plots with insecticide treatments in these 
months (Fig. 8). The first and second applications of dimethoate was followed by the 
increase of nymph colonies in both orchards, although following applications of this 
insecticide resulted in the decrease of both adults and nymph colonies for less than two 
weeks after the application (Fig. 8 A, D). Adults in the plot with imidacloprid treatment 



Differential Efficacy of Insecticides According to Crop Growth: The Citrus Psyllid on Citrus Plants 45 



maintained low densities throughout this experiment in CB1. Distinct reduction of nymph 
colonies by imidacloprid was seen only in August, however, and nymph colonies rather 
increased in the other months (Fig. 8B, E). The application of the mixture of these two 
insecticides resulted in decreases of both adult and nymph psyllids throughout this 
experiment, except for the nymph in July in CB2. 

The effects of insecticide treatment on the psyllid was evaluated by MANOVA, in which the 
means of the counts of adult and nymph psyllids per sampling time were culculated for 
individual trees and compared among orchards and treatments. The numbers were 
significantly different between orchards (F2, 98 = 23.016, P < 0.001) but not between 
treatments (Fe,i% = 2.058, P = 0.060) .The interaction of orchard and treatment was significant 
(F 6 ,i96 = 3.272, P = 0.004). 



(A) 



a 



Dimethoate 

5 Orchard CB1 
5 Orchard CB2 




:-*5T>*0 



(D) 



Dimethoate 

5 Orchard CB1 
£ Orchard CB2 




JullO Sep 10 Nov 10 

Sampling time (m/y) 

(B) , 

Imidacloprid 

4 $ Orchard CB1 

— . <£ Orchard CB2 



5 j ]f V V V ^ 



Jul 10 Sep 10 Nov 10 

Sampling time (m/y) 




Sep 10 
Sampling time (m/y) 



(C) 



Dimethoate + Imidacloprid 

5 Orchard CB1 
5 Orchard CB2 



JullO Sep 10 Nov 10 

Sampling time (m/y) 



Dimethoate + Imidacloprid 





JullO Sep 10 Nov 10 

Sampling time (m/y) 



JullO Sep 10 Nov 10 

Sampling time (m/y) 



Fig. 8. Densities of adults (A-C) and nymph colonies (D-F) in plots treated by either 
dimethoate 0.40 g/tree, imidacloprid 0.20 g/tree or the mixture of these two insecticides in 
two orchards, where seedlings had been planted either in May 2007 (CB1) or in November 
2007 (CB2). Arrows show the time when insecticides were sprayed. Bars indicate the 
standard errors of the means. 



46 



Pesticides in the Modern World - Risks and Benefits 



Any insecticide did not show long residue effects in this experiment. Furthermore, psyllids 
were increased in plots with insecticide treatments when few or no psyllids were found in 
the control plot. In particular, psyllids were likely to be more in insecticide-treated plots 
after September 2010 than in the control plot, even though psyllids were reduced just after 
the insecticide application. The interative factors in the increase in these plots are still 
unknown. One possible explanation seems to be predators of the psyllid that had been 
eliminated by insecticide application and their populations could not be recovered soon. 
Other interesting results were the difference in the the efficacy of imidacloprid on adults 
and on nymph colonies. Adults were well controlled by imidaclprid, but nymphs appreared 
to be free of the insecticide effects. However, adults in the plot with the treatment of the 
mixture of two insecticides were as many as those in the plot with only dimethoate. In the 
experiment of methidation application in field, imidaclprid did not show such a control 
effect on either the adult or the nymph. The few occurrences of adults in this experiment 
seem to have been involved in other confounding factors. Furthermore, adult psyllids were 
likely to be more in CB2 than in CB1, although no apparent differences in the nymph 
numbers were seen. Thus, any insecticide effectively reduced psyllid populations just after 
the application, but the residue effect could not continue for longer than two weeks. 



2.5.2 Insecticide efficacy on the aphid and scale insect 

In the control plot, the increase of aphids was not necessarily correspondent between two 
orchards (Fig. 9A). In each orchard, only one increase was observed: in September in CB1 
and in July in CB2. On the other hand, scale insects increased similarly in these two 
orchards: one increase in August and the other in September to October (Fig. 9B). These 
densities were compared with those in plots with insecticide treatments. Generally, aphid 
populations after insecticide application were reduced in both orchards, but increased in 
one to two weeks later (Fig. 10A, B). In October to November, their populations were 
recovered in the plots with dimethoate or imidacloprid treatment in two weeks after the 
application. Although scale insects were found more in CB2 than in CB1, poulation densities 
of the scale insect were likely to be higher in the plots with the treatment by only 
imidacloprid in both orchards (Fig. IOC, D). Application of dimethoate effectively 
suppressed the scale population, but imidacloprid led the scale rather to increase more than 
the no-insecticide treatment. The population of scale insects were particularly higher in CB2 



(A) 



(B)l.o r 



Kpbid 

5 Orchard CB1 
2 Orchard CB2 



Scale insect 

5 Orchard CB1 
<£ Orchard CB2 




+1W 




Sep 10 Nov 10 

Sampling time (m/y) 



JullO Sep 10 Nov 10 

Sampling time (m/y) 



Fig. 9. Densities of aphid (A) and scale insect colonies (B) in plots without insecticide treatment 
for the control of psyllids in two orchards, where seedlings had been planted either in May 
2007 (CB1) or in November 2007 (CB2). Bars indicate the standard errors of the means. 



Differential Efficacy of Insecticides According to Crop Growth: The Citrus Psyllid on Citrus Plants 47 



during the late August to early October than in the control plots. In the plots with the 
mixture of both insecticide, populations were maintained lower than the others. 
Univariate ANOVA was used to compare the effects of insecticide treatments on the aphid 
or scale insect population. The aphid population density was significantly different between 
treatments (F 3 , 99 = 4.078, P = 0.009) but not between orchards (F a , 99 = 0.253, P > 0.05). The 
interaction of these two variables was not significant (F3, 99 = 0.319, P > 0.05). The population 
density of scale insects was significantly different both between orchards (Fi, 99 = 6.206, P = 
0.014) and between treatments (F 3/ 99 = 3.443, P = 0.020). The interaction of orchard and 
treatment was not significant (F3, 99 = 0.506, P > 0.05). These results indicate that aphids 
could be effectively controlled by either dimethoate or imidacloprid but the efficacy would 
be continued for two weeks at longest. Imidacloprid would more effectively control aphids 
than dimethoate. Scale insects would be controlled effectively by dimethoate, but 
imidacloprid should be avoided for the control of the scale. Hence, when aphids are 
dominant and scales insects were few, either dimethoate or imidacloprid could be used. 
However, when scales were abundant, imidaclprid should be avoided even for the control 
of aphids, and dimethoate should be selected. If both pests are abundant, the mixture of 
these insecticides would be considered. It should be noted that the residue effects in any 
insecticide application would continue for two weeks at longest. 



(A) 



Orchard CB1 

- 5 Dimethoale 
5 Imidacloprid 
5 Dimethoale i Imidacloprid 




(B) 







Orchard CB2 

U Dnnclhoate 
U Imidacloprid 
5; Dimethoate 1 Imklaefoprid 


h 


} 


4 4 


i i 


1 


1 


> A-Tiu A-fii. 


ytt 



Sep 10 
Sampling time 



(in/>) 



JullO Sep 10 Nov 10 

Sampling time (m/y) 



(C) 



OrhcardCBl 

J Dimethoate 
J Imidacloprid 
5 Dimethoale Hmklac fop rid 






(U) 




Orchard CB2 
5 Dimethoate 
5: Imidacloprid 

2 Dimethoate I ItnidLickipnJ 



* )'i i i 

?CO? A ii ~ ^llS -<IJ- 1— J-; <li s Vi> 



Sep 10 Nov 10 

Sampling time (/tree) 



Jul 10 Sep 10 Nov 10 

Sampling time (m/y) 



Fig. 10. Densities of aphid (A, B) and scale insect (C, D) colonies in two orchards, where 
seedlings had been planted either in May 2007 (CB1) or in November 2007 (CB2). Each 
orchard was divided into four plots, to each of which any of the four treatments were 
randomly distributed: no insecticide use for the control of psyllids, 0.40 g/tree dimethoate 
by spray, 0.20 g/tree imidacloprid by spray, or 0.40 g dimethoate and 0.20 g 
imidacloprid/tree by spray. Arrows indicate the time when insecticides were sprayed. Bars 
show the standard errors of the means. 



48 Pesticides in the Modern World - Risks and Benefits 

3. Conclusion 

When trees are young, usually until one and a half years after planting in southern Vietnam, 
neonicotinoids by soil drench are expected to control psyllids effectively for two months 
after the application. Methidathion by spray can keep high residue effects for a half month 
for the control of psyllids on seedlings. This insecticide can quickly kill psyllids in 12 hr after 
their infestation on plants, and neonicotinoids did not attain such quick effect on the psyllid. 
This is the advantage in the use of methidathion. Dimethoate showed similar lethal effects 
on the psyllids as methidathion, although both its quickness and residue effects were 
inferior to those of methidathion. Field application of imidacloprid, methidathion, 
dimethoate, and the mixture of imidacloprid with any of the two organophosphates showed 
that these insecticides showed similar effects on the control of psyllids: their residue effect 
was maintained for less than two weeks and no insecticides succeeded in attained high 
lethal effect to eradicate psyllids on trees even in a couple of days after the spray. It should 
be noted that dimethoate could not control aphids as much as imidacloprid or its mixture 
with dimethoate but imidacloprid would lead the increase of scale insects after the 
application. The application of any insecticide examined in this study did not lead these 
pests to be eradicated for even one week after the application. These results indicate that the 
application of insecticides cannot be expected to attain perfect protection of citrus trees from 
CG infection once the tree grew. Furthermore, since nymphs could increase in two weeks 
after the application without elimination, even secondary infection would not be avoided 
when citrus trees have grown to a stage of fruit yielding. The application of insecticide 
would only reduce more or less the probability of the second infection of trees by CG in the 
orchard. 

4. Acknowledgment 

This study was performed under the collaborative research project, no. 3241, of Japan 
International Research Centre for Agricultural Sciences (JIRCAS) with the Southern 
Horticultural Research Institute of Vietnam (SOFRI). Prof. Emeritus Su, H.-J. at the Taiwan 
University provided us with precious suggestions in the study. Dr. Chau, N. M. supported 
our studies both officially and scientifically. Dr. Koizumi, M. and Dr. Kano, T. suggested us 
about works on citrus both in Japan and other countries. Dr. Yonemoto, M. and Mr. Ogata, 
T. at JIRCAS provided information necessary for citrus cultivation. Dr. Hoa, N. V. and Mr. 
Dien, L. Q. gave us information of citrus in Vietnam. Miss Nga, V. T. and Miss Oanh, T.T.K. 
helped our works both in laboratory and field. We would like to express our sincere thanks 
to them. 

5. References 

Boina, D.R.; Onagbola, E.O., Salyani, M. & Stelinski, L.L. (2009). Antifeedant and sublethal 
effects of imidacloprid on Asian citrus psyllid, Diaphorina citri. Pest Management 
Science, 65, 870-877. 

Gatineau, F.; Bonnot, F., Yen, T. T. Hong, Tuan, D. H, Tuyen, N. D., & True, N. T. N. (2010). 
Effects of imidacloprid and fenobucarb on the dynamics of the psyllid Diaphorina 



Differential Efficacy of Insecticides According to Crop Growth: The Citrus Psyllid on Citrus Plants 49 

citri Kuwayama and on the incidence of Candidatus Liberibacter asiaticus. Fruits, 

Vol.65, pp. 209-220 
Halbert, S.E. & Manjunath K.L. (2004). Asian citrus psyllids (Sternorhyncha: Psyllidae) and 

greening disease of citrus: a literature review and assessment of risk in Florida. 

Florida Entomologist, Vol.87, pp. 330-353. 
Hayashikawa, S., Suenaga H. & Torigoe H. (2006). Insecticidal activity of some insecticides 

on Asian citrus psyllid, Diaphorina citri Kuwayama. Kyushu Plant Protection 

Research, 52, 71-74. (In Japanese with an English summary) 
Hung, T.-H.; Hung S.-C, Chen C.-N., Hsu M.-H. & Su H.-J. (2004). Detection by PCR of 

Candidatus Liberibacter asiaticus, the bacterium causing citrus huanglongbing in 

vector psyllids: application to the study of vector-pathogen relationships. Plant 

Pathology, Vol.53, pp. 96-102. 
Ichinose, K.; Bang, D.V., Tuan, D.H. & Dien, L.Q. (2010a). Effective use of neonicotinoids for 

protection of citrus seedlings from invasion by Diaphorina citri (Hemiptera: 

Psyllidae). Journal of Economic Entomology, Vol.103, pp.127-35 
Ichinose, K. & Kano, T. (2006). Citrus greening disease and its management in the Mekong 

Delta Region of Vietnam. Shokubutsuboeki, Vol.60, 302-307. (In Japanese) 
Ichinose, K.; Miyaji, K., Matsuhira, K., Yasuda, K., Sadoyama, Y., Tuan, D.H. & Bang, DV. 

(2010b). Unreliable pesticide control of the vector psyllid Diaphorina citri 

(Hemiptera: Psyllidae) for the reduction of microorganism disease transmission. 

Journal of Environmental Science and Health Part B, Vol.45, pp.466-472. 
Inoue, H; Ohnishi, J., Ito, T., Tomimura, K., Miyata, S., Iwanami, T. & Ashihara, W. (2009). 

Enhanced proliferation and efficient transmission of Candidatus Liberibacter 

asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage. 

Annals of Applied Biology, Vol.155, pp. 29-36. 
Koizumi, M.; Prommintara, M., Linwattana, G. & Kaisuwan, T. (1997). Epidemiological 

aspects of citrus huanglongbing (greening) disease in Thailand. JARC, Vol.31, pp. 

205-211. 
Quresh, J.A.; Stansly, P.A. (2010). Dormant season foliar sprays of broad-spectrum 

insecticides: An effective component of integrated management for Diaphorina citri 

(Hemiptera: Psyllidae) in citrus orchards. Crop Protection, Vol.29, pp. 860-866. 
Srinivasan, R., Hoy, M.A., Singh, R. & Rogers, M.E. (2008). Laboratory and field evaluation 

of Silwet L-77 and kinetic alone and in combination with imidacloprid and 

abamectin for the management of the Asian citrus psyllid, Diaphorina citri 

(Hemiptera: Psyllidae). Florida Entomologist, Vol. 91, pp. 87-100. 
Yang, Y.; Huang, M., Beattie, G. A. C, Xia, Y., Ouyang, G. & Xiong, J. (2006). Distribution, 

biology, ecology and control of the psyllid Diaphorina citri Kuwayama, a major pest 

of citrus: a status report for China. International Journal of Pest Management, Vol.52, 

pp. 343-352. 
Yasuda, K; Ooishi, T. & Kawamura, F. (2006). Effect of insecticides on adults and larvae of 

Asian citrus psyllid, Diaphorina citri (Homoptera: Psyllidae). Kyushu Plant Protection 

Research, Vol.52, pp. 75-78. (In Japanese with an English summary) 
Yasuda K.; Yoshitake H, Ooishi T., Toudou A. & Uechi N. (2007). Effect of high-density 

scatter of the infiltration shift insecticide on invasion by adult and egg-laying 



50 Pesticides in the Modern World - Risks and Benefits 

individuals of the Asian citrus psyllid, Diaphorina citri (Homoptera: Psyllidae). 
Kyushu Plant Protection Research, Vol.53, pp.95-98. (In Japanese with an English 
summary). 



Use of Pesticides in the Cocoa 
Industry and Their Impact on the 
Environment and the Food Chain 

George Afrane 1 and Augustine Ntiamoah 2 

University of Ghana, Department of Food Process Engineering, 

2 Koforidua Polytechnic, Department of Energy Systems Engineering, 

Ghana 



1. Introduction 

Cocoa, Theobroma cacao L., is a major cash crop cultivated in the tropical regions of West 
Africa, the Caribbean, South America and Asia. In West Africa, where over 70% of the 
world's cocoa is produced - with about 21% coming from Ghana - it is a significant 
component of the rural economy, as the industry is dominated by large numbers of small- 
holder peasant farmers who depend on the crop for their livelihood (Acquaah, 1999; 
Appiah, 2004). Like all living organisms, the cocoa plant can also be attacked by a wide 
range of pests and diseases. When this happens expected production targets are not met, 
and the economies of the producer nations are adversely affected. Preventive and curative 
measures are therefore necessary in the cocoa industry to maintain and even increase output 
(Akrofi and Baah, 2007). 

While non-chemical means of managing pests and diseases in the industry are widely 
recommended for health and other reasons, the use of some amounts of chemicals in the 
form of fertilizers, insecticides and fungicides is unavoidable in the effective management of 
cocoa farms (Moy and Wessel, 2000; Opoku et al., 2007; Adjinah and Opoku, 2010). Their use 
is therefore expected to increase with time. Indeed in the twenty-year period from 1986- 
2006, the use of fertilizer world-wide increased by almost 250% (UNEP, 1991). The same 
trend applies to pesticides, although they are more difficult to monitor partly because of the 
secrecy that goes with the continued production and use of banned substances. The trends 
suggest quite clearly however, that much of the increase in world food production can be 
attributed to the response of crops to increased use of fertilizers and pesticides (UNEP, 
1991). Fortunately, there has always been a clear appreciation of the potential deleterious 
effects of the chemicals used in the cocoa industry since the 60s, and standards have been set 
by FAO and WHO for acceptable levels of residues in the beans exported to other countries. 
The goal of maintaining high levels of agricultural productivity and profitability while 
reducing pesticides use presents a significant challenge. There are repeated cases of 
excessive levels of pesticide residues being found in agricultural produce and the safety of 
these products has become an issue of concern. Recently, changes in regulations in the 
European Union (EU), North America and Japan have called for a reflection on crop 
protection practices in cocoa and other commodity crops (ICCO, 2007). The quality of cocoa 



52 Pesticides in the Modern World - Risks and Benefits 

imported into the EU and elsewhere will be assessed based on traces of pesticides and other 
substances that have been used in the supply chain. 

The cocoa bean has a high content of butter or fat which absorbs the active ingredients in 
insecticides. The acceptable levels of active ingredients in foods are determined by the 
committee on Pesticide Residue of FAO/WHO, known as the Codex Alimentarius 
Commission, CAC. Created in 1963 the CAC implements the Joint FAO/WHO Food 
Standards Programme which is aimed at protecting the health of consumers and ensuring 
fair trade practices in the international food trade (Moy and Wessel, 2000). The commission 
has set maximum levels of residue poisons in commodities going through the international 
market, including cocoa. If for any reason the residual levels in any commodity exceed the 
Codex levels, that particular commodity could be rejected by the importing country. 
Secondly, the accumulation of any chemicals in the cocoa fat may change the taste of the 
beans and eventually that of the chocolate made from them. This is known as tainting. It is 
therefore, the task of entomologists to ensure that recommended chemicals do not leave any 
residues, and that the dosage is the minimum that would give the optimum control under 
the agricultural conditions in the country. 

In Ghana, significant gains have been made in the control of pests and diseases of the cocoa 
industry through the nationwide use of pesticides under government sponsorship and 
supervision. The growing global concerns about the effects of the increasing use of 
agricultural chemicals on farmers, consumers of agricultural produce and the ecology 
require a re-examination of the issues related to their application in the cocoa industry. This 
chapter examines the use and the impact of pesticides in cocoa production in Ghana - where 
data has been accumulated - as a representative country of the industry. The potential 
ecological impacts of chemicals in the cocoa industry are analyzed, using the modern tool of 
life-cycle assessment (Ntiamoah and Afrane, 2009). Life-cycle assessment, LCA, has gained 
such prominence in the environmental management discipline that the International 
Standards Organization has developed standards for its implementation (ISO 14040-14043, 
1997-2000b). This particular analysis is based on primary farm-level data collected from a 
nationally representative sample of cocoa farmers, published data, results from research 
institutions, the Ghana Cocoa Board and other relevant sources. 

2. The Ghanaian cocoa industry in brief 

In Ghana, cocoa has played an important role in the economy of the country for over one 
century. Although the crop was believed to have been brought to the colonial Gold Coast - 
as Ghana was then known - from Fernando Po, an island in the Gulf of Guinea, off the coast 
of Gabon, in 1879 and from Sao Tome in 1886, records show that in 1891, only twelve years 
after it first arrived here, cocoa was being exported as a cash crop (Acquaah, 1999, Adjinah 
and Opoku, 2010). From the 1910/1911 season, Ghana became the leading cocoa producer in 
the world, a position it held until 1977, when it was overtaken by the Ivory Coast. The 
country went from being the number one cocoa producer to a period in the early 80s when, 
as a result of drought, bushfires, low producer prices, diseases and general economic 
malaise, Ghana fell to the twelfth position and produced less than 160,000 metric tonnes in 
the 1983/1984 season (Adjinah and Opoku, 2010). 

Cocoa became attractive as a cash crop in Ghana because of the lower cost involved in its 
cultivation, compared to a popular crop like palm, as well as the favourable natural 
conditions that existed in the forest belts. Cocoa could be grown along with other crops and 



Use of Pesticides in the Cocoa Industry and Their Impact on the Environment and the Food Chain 53 

when soil conditions deteriorated the land could be left to the cocoa trees and other tracts 
tilled in the shifting-cultivation systems of farming (Acquaah, 1999). Because of the 
prominence that the crop had began to gain in the economy, even before World War II, 
government was seriously alarmed when the swollen shoot disease was discovered in 1936. 
In the process of combating this disease, a permanent research center was established at 
Tafo, in the Eastern Region, and product quality inspectorate, grading of beans, extension 
services and proper engagement of farmers in the growth of the crop were initiated 
(Acquaah, 1999). Since then government has continued to offer technical assistance, financial 
incentives and inputs like fertilizer and pesticides to cocoa farmers. 

Over the last decade, as a result of government intervention, cocoa production has picked 
up, reaching a peak of 740 thousand metric tonnes in the 2005/2006 season (Aryeetey et al, 
2007). Constituting 7.3% of the Gross Domestic Product of the country, it is second only to 
gold, which first overtook cocoa as the highest foreign exchange earner in 1992; a trend 
which still continues. Agriculture contributes about 35% of Ghana's Gross Domestic Product 
(GDP) and 60% of total employment. The Cocoa Industry is the single largest contributor to 
agricultural GDP (16.5 %). It is estimated that about 65% of the country's agricultural 
workforce work either directly or indirectly in the cocoa industry. In Ghana cocoa is grown 
on small farms owned by individuals and families in the forest zones of Ashanti, Brong 
Ahafo, Western, Eastern and Volta regions. Thus the livelihood of about two million farmers 
and their dependants, mostly in the rural areas, depend directly on cocoa (Opoku et. al, 
2006). 

2.1 Cocoa processing in Ghana 

Although serious attempts have been made to process them locally, the majority of cocoa 
beans produced in the country are still exported. Government put a policy in place to 
process at least 50% by the end of the last decade. The enabling conditions created in free 
zone enclaves, led to the attraction of private foreign processing companies and the 
expansion of state-owned facilities. According to data from International Cocoa 
Organization, ICCO, 200,000 metric tonnes of cocoa grindings were achieved in Ghana in 
the 2009/2010 season. Compared to the production figure given in Table 1 for the same 
season, this constitutes about 32% of the beans produced. This means the government's 
target for grindings was not achieved. 

In spite of their peripheral role in the standard household menu - mainly as a dessert or 
snack, food products made from cocoa go through a long line of operations not normally 
found with other processed foods, as depicted in Figure 1 (Awua, 2002). Ripe cocoa pods are 
plucked from the trees and gathered together on clearings in the cocoa farms. After about 
ten days, all available hands, young and old, gather together to assist in the splitting of the 
pods and removal of the beans with their hands. (According to Owusu-Manu (1977), this 
could be a critical stage in the contamination process, with pesticides getting transferred 
from the workers to the wet beans.) The wet beans are collected together in a heap and 
covered with plantain leaves and plastic sheets for fermentation. After fermentation, the 
beans are dried in the sun on bamboo mats to a desired moisture content of around 7.5%. 
After dried cocoa beans have been received at the processing plant, they are inspected and 
thoroughly cleaned of all extraneous matter, such as sticks, stones, metal fragments, dust, 
loose shells, small fragments and clumps of cocoa beans. The cleaning process consists of a 
series of operations involving sieves, brushes, airlifts and magnetic separators to remove the 



54 



Pesticides in the Modern World - Risks and Benefits 























Cocoa production 










i Jute plant cultivation 






i 








T" 


Fertilizer production 






Fermentation and 
Drying 








i Jute bag Manufacturing 


















Pesticides production 






1 






T 








Bagging and Storage 






1 






Industrial cleaning of 
beans 






I 








Roasting 






i - - - - - - -i 

1 Cattle Raising | 


Sugar beet cultivation 




1 




"V 




T 

Winnowing 




; 




r i 

i Sugar production 
i 


i Milk production j 




i 
















Grinding 




















♦ 1 t 










Mixing of cocoa liquor, 

milk, sugar and other 

ingredients 




PE production [ 






1 






"1 




Conching and Refining 


] Paper box packaging ' 




Film packaging 
i production 


1 


production 






Tempering and Moulding 
























+ 


- ♦ 








Chocolate packaging 
1 




i 
i 

! 

1 


1 


i 
i 


Distribution and Retail 


1 


Consumption phase 

i 


1 


] Expired food & 
packaging disposal 



Fig. 1. Process Flowchart for Chocolate Production 



Use of Pesticides in the Cocoa Industry and Their Impact on the Environment and the Food Chain 55 

unwanted materials. The cleansed cocoa beans are roasted at temperatures between 90- 
170°C, using a petroleum-based fuel or electricity. This process is needed to develop the 
chocolate flavour, reduce the moisture content further, and loosen the shells for subsequent 
removal. The nibs (cotyledons) become friable and generally darker in colour in the process. 
At the next stage, the shells are separated from the nibs in a process known as winnowing. 
Winnowing machines use a multi-layered sieve frame with meshes of different sizes, one 
above the other, with the largest mesh on top. The roasted and crushed beans are ground 
into a paste known as cocoa liquor or cocoa masse. The grinding process is achieved in two 
or three stages, using a combination of mills. The cocoa liquor obtained is heat-treated in 
storage tanks at temperatures of between 90-100°C for aging and microbial destruction. 
The cocoa paste could be pressed in a hydraulic device to extract cocoa butter. The cake 
released after pressing is passed through kibbling machines, which break them into smaller 
pieces, and are packed into four-ply multi-walled paper sacks lined with polyethylene. 
These are ready for sale and shipment as kibbled cake. The cocoa butter, on the other hand, 
may be mixed with the other ingredients of chocolate, namely, butter, sugar, milk and 
emulsifiers. The chocolate mix is subjected to additional processes known as conching and 
tempering. Conching removes residual moisture, while tempering transforms the thick 
semi-liquid mix into a solid product through heat treatment. After this process the chocolate 
is poured into moulds of different shapes and then packaged for the market. Knowledge of 
the material and energy requirements of each of the processes as shown in figure 1 is 
necessary to perform the LCA analysis needed to determine the environments impacts. 

2.2 The place of cocoa in the food chain 

While the soporific effect of cocoa drinks is widely known, recent research activities have 
unearthed additional more important health benefits which have enhanced further the 
attractiveness of cocoa products generally. There are three types of chocolate: dark, milk and 
white chocolates. Most of the benefits of chocolate consumption are associated with the dark 
brand. In the last decade, studies have shown that chocolate consumption can play an 
important role in the reduction of risks or delaying the development of cardiovascular 
diseases, cancer and other age-related diseases. It has also been linked positively to anti- 
carcinogenic activity in human cells, hypertension, diabetes and sexual weakness. It's newly 
found reputation as an aphrodisiac, stems from the ability of its sweet and fatty nature to 
simulate the hypothalamus, which induces pleasure sensation and affects the level of 
serotonin in the brain (Afoakwa, 2008). 

Cocoa products contain flavonoids and amino acids, and these have been cited as the source 
of its beneficial effects, while carbohydrates, theobromine and lead have been mentioned as 
responsible for the negative effects. The flavonoids belong to a large and complex group of 
compounds called polyphenols and are found in plant products, mainly fruits and 
vegetables. The phenols in cocoa products have been associated with antioxidant properties, 
reduction in migraine, protection of arteries from plaque formation and prevention of LDL 
formation two hours after consuming dark chocolate and perceptible lowering of blood 
pressure. Some studies have also linked chocolate consumption to muscle recovery and 
delayed brain function decline (Reuters, 2007). Protein is broken down in the body to form 
twenty amino acids needed by the body. Eight of these are called essential, which means 
they are not made by the human body itself and must be supplied from outside. Fourteen of 
the twenty amino acids found in the body, including the eight essential ones, have been 



56 Pesticides in the Modern World - Risks and Benefits 

found in cocoa. In addition to building cells and repairing tissues, amino acids also have 
antioxidant properties, and they form antibodies to combat invading bacteria and viruses 
(Awuah, 2002). 

While international standards are such that the pesticides used in the field can hardly find 
their way into chocolate, a number of documented negative effects have been associated 
with some of the natural and absorbed constituents of cocoa. Perhaps the major one is 
obesity. It is believed that the amounts of dark chocolate that needs to be consumed in order 
to experience the good benefits of the product could lead to obesity and its resultant 
negative effects. Although it is not supported by scientific studies, it is also believed that 
chocolate consumption can lead to acne (www.chocolate.gourmetrecipe.com). The heavy metal, 
lead, is known to maintain a high solubility in chocolate, and this may lead to lead 
poisoning (Rankin et al, 2005). Chocolate is also known to be toxic to some animals like 
horses, dogs, parrots, cats and small rodents, because they are unable to metabolize the 
theobromine which is found in chocolate (Drolet et al, 1984; Blakemore and Shearer, 1943). 

3. Pests and diseases of cocoa 

The increasing world population cannot be sustained without the use of pesticides in food 
production. Their usage therefore benefits not only farmers but also consumers. Pesticides 
are used to reduce food losses not only during production, but also during the post-harvest 
storage stage (Moy and Wessel, 2000). The general pest control strategy is for the 
intervention to destroy the pests feasting on the crops, but at the same time not to damage 
the produce so much as to render them unhealthy or unprofitable. This means looking for 
the thin line which separates good practices from bad. Good agricultural practice (GAP) 
requires good timing and proper application. The crops are sprayed on the advice of 
specialists at an opportune time in the reproductive cycle of the pest, when the highest 
numbers could be eliminated. Also in order to maintain the activities of friendly insects the 
area of application of the insecticides should be clearly delineated. 

The cocoa tree and its pod can be attacked by different species of insects, fungal diseases 
and rodents (Entwistle, 1972). The major diseases affecting cocoa in Ghana are given in 
Table 1. The most important of these are Phytophthora pod rot, commonly called "black 
pod", and locally known as 'akate'; and the swollen shoot virus, also known locally as 'cocoa 
sasabro'. The black pod rot, a fungal disease which appears as characteristic brown necrotic 
lesions on the pod's surface and as rotting of the beans, does the most damage to cocoa. An 
estimated 30% of annual cocoa production is lost to it, especially during years of high 
rainfall. At 2005 cocoa bean prices this is an estimated US$1.5 billion in lost revenue 
(www.icco.org). Other estimates put the loss specifically at 450 thousand metric tonnes 
annually, while 250, 200 and 50 thousand MT are lost to witches' broom, capsids, and the 
swollen shoot virus (CSSV), respectively (www.dropdata.org). Witches' broom and frosty pod 
rot are predominant in Latin America, while the black pod and CSSV are common in West 
Africa. These diseases are counted by breeding disease-resistance species, sanitation and the 
use of fungicides (Bastos, 1996; Opoku et al, 2007). 

Most insects which attack cocoa are of the bug or miridiae family. This is a large family of 
insects of which capsids, the most well-known, have achieved their notoriety from the 
degree of havoc they can wreck on cash crops like cocoa. They feed on plants by piercing the 
tissue and sucking their juices. Capsids are small, terrestrial insects, usually oval-shaped or 
elongate and measuring less than 12 mm. They were identified as pests at the turn of the last 
century and are the main insects that feed on cocoa in Africa (Mahot et al., 2005). 



Use of Pesticides in the Cocoa Industry and Their Impact on the Environment and the Food Chain 57 





Disease 


Type of Infection 
(Causal agent) 


Symptoms 


Black pod 


Fungus 
(Phytophthora spp.) 


Pod rots, go brownish-black. Beans destroyed 
in immature pods. Could result in die-back 


Brown root rot 


Fungus 

(Forties noxius) 


Leaves fall prematurely and die-back of twigs 
occurs. Fungus fruit bodies on root and dead 
trunks. Soil is affected 


Cocoa necrosis 


Virus 

(Cocoa necrosis virus) 


Leaves show bands of transparent lesions 
often with perforated centers 


Collar crack 


Fungus 
(Armillaria mellea) 


Longitudinal cracking of trunk from ground 
level to about 1.2m upwards, fills with 
cream-coloured mycelium 


Collar rot 


Fungus 
(Ustulina zonata) 


Defoliation and death of plants. White fan- 
shaped patches of mycelium are produced 
underneath bark and roots 


Cushion gall 


Fungus 

(Calonectria rigidiuscula) 


Excessive production of buds at the nodes 


Vascular 

Streak 

Die-back 


Fungus 

(Oncobasidium theobroma) 


Leaves turn yellow and fall prematurely. 
Smaller branches wither starting from the 
tips 


Horse hair 
blight 


Fungus 

(Marasmius equicrinis) 


Network of black threads which spread 
throughout the canopy, smothers shoots 
growth 


Mealy pod 


Fungus 

(Trachysphaera fructigena) 


Pods turn brown, becomes encrusted with 
white to pinkish mealy growth of the fungus 


Mistletoe 


Flowering Plant 

(Tapinanthus bangwensis) 


Parasitic flowering plant on host branches. 
Part of branch withers 


Pod rot 


Fungus 

(Botryodiphlodia 

theobromae) 


Appears as brown necrotic areas with 
concentric rings of black spots. Pods are later 
covered with black sooty powder 


Red rust 


Alga 

(Cephaleuros mycoidea) 


Reddish patches on leaves and twigs; leaves 
are shed prematurely 


Swollen shoot 


Virus 

(Cocoa swollen shoot 

virus) 


Swelling of chupons and twigs; leaves 
develop yellow patterns, get crinkled and 
malformed 


White Root 


Fungus 
(Fomes lignosus) 


Premature defoliation, death of twigs, pods 
are small 


White thread 
Blight 


Fungus 

(Marasmius scandens) 


Leaves are covered and killed in a network of 
white mycelial threads 



Source: Offei et al. (2005) 

Table 1. Diseases of Cocoa in Ghana 



3.1 National cocoa pests and diseases control programme 

Throughout the 90's, the tonnage of cocoa produced annually rarely exceeded 400,000 
metric tonnes. This situation was attributed to a variety of causes, although the prevalence 
of pests and cocoa diseases was seen as the main reason. Crop losses due to mirids alone 



58 



Pesticides in the Modern World - Risks and Benefits 



were estimated at between 25-35% per annum. To reverse this trend, the government of 
Ghana in the year 2000 introduced the national Cocoa Diseases and Pests Control 
Programme, CODAPEC, popularly known as "mass spraying", to combat the resurgence of 
mirids and black pod diseases on cocoa farms. This opportunity was also to be used to train 
farmers and technical personnel in the scientific methods of pests and diseases control 
(Adjinah and Opoku, 2010). Participants were trained in the dosage of the various 
pesticides, dangers of exposure to pesticides, importance of the use of protective clothing, 
observance of personal hygiene, environmental safety issues, first-aid, techniques of 
application and handling and disposal of empty containers. Lessons were given through 
radio programmes, town meetings and 'training-of-trainers' workshops. Table 2 gives the 
brands of pesticides, approved by the Cocoa Research Institute of Ghana (CRIG), which are 
currently in use on Ghanaian cocoa farms under the CODAPEC programme and their 
application frequency. 



Pesticide used 


Active ingredient 


Method of 
application 


Frequency 


Fungicides 








Ridomil 72 plus 


12% metalaxyl, 60% 






WP 


Cuprous oxide 






Nordox 75 WP 


86% Cuprous oxide, 14% 
inert 






Funguran OH 
WP 


Cuprous hydroxide 


Knapsack 


3 times during 


Champion WP 


77% cupric hydroxide 


sprayer 


each cocoa season 


Kocide 101 WP 


Cupric hydroxide 






Fungikill WP 


Cupric hydroxide + 






Metalm 72 Plus 


metalaxyl 






WP 


Cuprous oxide + metalaxyl 






Insecticides 








Akatemaster 


Bifenthrin 






Actara 


Thiamethoxam 






Cocostar 210 EC 


Bifenthrin + Pirimiphos- 


Knapsack 


Twice during each 




methyl 


sprayer 


cocoa season 


Confidor 200SL 


Imidacloprid 






Carbamult 


Promecarb 







Table 2. Pesticides approved for used in the control of mirids and black pod disease under 
the CODAPEC programme 

The black pod control programme covered all cocoa-growing districts in the Volta, Brong 
Ahafo and parts of Western, Ashanti and Eastern Regions. Spraying against mirids, on the 
other hand, covered the Central, Eastern and parts of Western and Ashanti Regions. 
Spraying gangs were established at each spraying centre. A gang of ten (for black pod 
control) and six (for mirids control) had a supervisor each responsible for the general 
execution of the programme at the unit level. One mechanic was attached to a group of 20 
gangs to oversee the maintenance and repairs of the spraying machines. The farmers, who 



Use of Pesticides in the Cocoa Industry and Their Impact on the Environment and the Food Chain 59 

were direct beneficiaries of the exercise, were themselves responsible for the sanitation 
practices, i.e. brushing, pruning, shade management and removal of diseased pods from the 
farms. They also provided water for spraying and were expected to monitor the activities of 
the sprayers on the farm. The spraying is carefully done using a portable petrol-engine- 
driven knapsack mist-blowers, which combines the idea of low-volume application of 
sprays with the principle of using fan-driven air to carry the spray up into the trees. 
As a result of this initiative, between the period 2002-2004, nearly 600,000 ha involving 
about 360,942 farms and 330,121 individual farmers, were sprayed three times each season 
against the black pod diseases, while an estimated 826,141 ha involving 470,801 and 446,593 
farmers were sprayed twice each season in the mirids control exercise. From the 2001/2002 
season when beans output of 380,000 metric tons was recorded, production jumped to about 
500,000 metric tonnes in the 2002/2003 season and almost doubled in the 2003/2004 season 
to an all-time high of over 736,000 metric tonnes. 

Started ten years ago, the mass spraying exercise has now become a permanent fixture in all 
the 72 geographical districts in the cocoa-growing areas with the following breakdown: 
21districts for black pod spraying only, 35 districts for mirids only, and 16 for both 
programmes. District Task Forces (DTF) and Local Task Forces (LTF), have been formed in 
each operational district and local area, respectively. The DTF manages the project at the 
district level and is in charge of gang recruitment, storage, distribution of inputs and logistics 
and general supervision. The LTF on the other hand, handles project management at the 
village level and is responsible for the planning and execution of the programmes at that level. 
Table 3 gives the seasonal cocoa production figures along with the amounts of fertilizers and 
pesticides which have been used in Ghana in recent years. The table indicates clearly that 
cocoa production has increased significantly in the last decade, but it has been at the expense 
of more pesticides and fertilizers. Data obtained from COCOBOD indicate that fourteen 
different kinds of insecticides and fungicides have been used for spraying farms since the start 
of the mass spraying exercise. Even with the limited data provided, the increase in pesticide 
usage per unit weight of cocoa over the period is evident. The same trend applies to fertilizer 
usage. Serious attention must be paid to these trends beyond the normal concerns with 
maximum residue limits (MRLs) which international traders focus on. The impact of these 
prodigious amounts of chemicals used in cocoa production on the environment as a whole can 
be determined through life-cycle analyses (Ntiamoah and Afrane, 2009). 



Crop 
Season 


Cocoa 
Production 

(106 kg) 


Total" 

Fertilizer 

Used 

(106 kg) 


Pesticides Usage" 


Fertilizer 

used per 

MT 


Fungicides 

used per 

MT 


Insecticides 

(liters) 


Fungicides 
(MT) 


2004/05 


601.9 


- 


1023.6 


1120.0 


- 


1.86 


2005/06 


740.4 


- 


745.0 


759.4 


- 


1.03 


2006/07 


614.5 


70.1 


590.0 


1120.0 


0.11 


1.83 


2007/08 


729.0t 


55.8 


1020.0 


1290.0 


0.08 


1.77 


2008/09 


662.0t 


105.0 


1760.0 


1800.0 


0.16 


2.72 


2009/10 


632.0t 


130.0 


2300.0 


1997.7 


0.20 


3.16 



tSource: ICCO Quarterly Bulletin of Cocoa Statistics, Vol. XXXVI, No. 4, Cocoa year 2009/2010. Published: 30- 
11-2010; all others in this column from, The State of the Ghanaian Economy, 2007. "Source: COCOBOD, Ghana. 

Table 3. Seasonal Cocoa Production, Fertilizer and Insecticide Usage in Ghana, 2004-2010 



60 



Pesticides in the Modern World - Risks and Benefits 



While non-chemical means of managing cocoa pests and diseases are widely recommended, 
the need for agro-chemicals to manage cocoa pests and diseases is unavoidable and will 
continue for years to come. However, the effects of continued exposure of users of 
pesticides, environmental risks, issues of pest resistance and possible hazards for consumers 
require a re-examination of the benefits of pesticide application and the risks involved. 
Hence the introduction of Good Agricultural Practices (GAP) to considerably mitigate, if not 
eliminate, the problems associated with the excessive and unnecessary application of 
pesticides. High residue levels and tainting of the beans could lead to their rejection on the 
international market. Testing for residues is carried out following internationally agreed and 
validated methods (Moy and Wessel, 2000). Though some insecticide residues are 
sometimes found in the shells, they are hardly found in the nib which is used in chocolate 
manufacture. 

4. Socio-economic impacts of pesticides use on the cocoa industry 

In terms of output the CODAPEC programme was a tremendous success, because it was 
able to resuscitate cocoa production in Ghana. The country continues to benefit not only 
because of increased output, but also because of the high prices the crop is currently 
enjoying on the international market. Thus the benefits to the economy as a whole were 
obvious. What was not so obvious was the direct benefit to the cocoa farmers. 
In order to assess the impact of the programme on these farmers, Abankwa et. al (2010), 
conducted a study in a typical cocoa-growing district, Ahafo Ano South, located at the 
north-western section of the Ashanti Region of Ghana. The study found that while the 
farmers could not take their children to better basic schools, they were able to afford school 
uniforms and other basic educational needs for them. They also found that farmers were 
able and more willing to visit hospitals instead of self -medicating or using herbal treatment. 
The improvements brought about by the programme seemed to benefit more farmers with 
higher levels of education, the study showed. 

One poignant conclusion of the study was that, while the price of cocoa was reviewed 
upwards every year over the first five years of the programme, these increments did not 
translate into increased purchasing power of farmers. They were not able to afford assets 
like radios, televisions, mattresses and vehicles any better, five years after the programme 
was started. Table 4 gives the nominal and actual farmers' income over the period 2001- 
2005. While the nominal figures trend upwards annually as a result of the increases in cocoa 
price, the actual income (calculated using CPI of 1997 as base) goes down every year due to 
the effect of inflation. 



Year 


Consumer Price Index 


Nominal Income 


Actual Income 


2001 


216.4 


631.5 


294.6 


2002 


246.2 


679.9 


276.2 


2003 


311.8 


753.3 


241.6 


2004 


351.2 


805.5 


229.4 


2005 


404.3 


939.3 


232.3 



Table 4. Variations in Farmers' Income, 2001-2005 (Source: Abankwa et al. 2010) 



Use of Pesticides in the Cocoa Industry and Their Impact on the Environment and the Food Chain 61 

From the point of view of COCOBOD, the implementers of the programe, the mass spraying 
exercise has been a roaring success, because of the increased yield it has generated, the 
renewed enthusiasm for cocoa cultivation that it has awoken in farmers, and also because of 
the 60,000 direct jobs it has created for sprayers, supervisors and mechanics in the rural 
areas of the country (Adjinah and Opoku, 2010). According to the Seed Production Unit of 
COCOBOD, demand for planting materials has gone up significantly because new farms are 
been established and old ones rejuvenated (Adjinah and Opoku, 2010). Farmers now clearly 
see cocoa farming as a profitable venture, especially with the continued reduction in 
inflation and the general improvement in the economy. 

5. Potential ecological impacts of pesticides use in cocoa production 

A proper assessment of the effect of pesticides and other chemicals used during cocoa 
production and processing on the environment and human health, has to begin with an 
effective quantification of the chemicals released into the environment and their impact on 
various aspects of human life and the environment. For this purpose, one of the widely 
accepted modern methods for examining the environmental impacts associated with a 
service or a product is the life cycle assessment (LCA) technique. 



5.1 Life cycle assessment methodology 

The Society of Environmental Toxicology and Chemistry (SETAC), defines LCA as: 

"an objective process to evaluate the environmental burdens associated with a product, 

process or activity by identifying and quantifying energy and materials used and wastes 

released to the environment; to assess the impact of those energy and material uses and 

releases to the environment; and to identify and evaluate opportunities to effect environmental 

improvements. The assessment includes the entire life cycle of a product, process or activity, 

encompassing extracting and processing raw materials; manufacturing, transportation and 

distribution; use, re-use, maintenance; recycling, and disposal" (Consoli et al, 1993). 

The International Organization for Standardization (ISO) has also provided very relevant 

input to the definition of LCA. According to ISO 14040 (1997), LCA is 

"a compilation and evaluation of the inputs, outputs and the potential environmental impacts 

of a product system throughout its life cycle. A product system is a collection of materially or 

energetically connected unit processes, which performs one or more defined functions". 



Goal and scope 

definition 

v. J 


1 


It 




( \ 
Inventory 

analysis 

v J 




1 




c > 

Impact 

Assessment 

v J 


\ 



Interpretation 



Fig. 2. Components of a Life Cycle Assessment (ISO 14040) 



62 Pesticides in the Modern World - Risks and Benefits 

The standard LCA methodology consists of four stages, namely, goal and scope definition, 
inventory analysis, impact assessment and interpretation of results. These are represented 
pictorially in Figure 2. The goal and scope definition means a clear statement of the reasons 
for performing the study, the intended use of the results and the specification of the basic 
parameters of LCA study, such as the functional unit, system boundaries, allocation rules, 
data quality and simplifications. According to ISO 14040:1997, the functional unit is defined 
as 'the quantified performance of a product system for use as a reference unit in an LCA 
study'. For a product this usually simply involves specifying the weight, volume or number 
of a unit amount. Thus it has to be clearly defined and measurable. The primary purpose of 
the functional unit is to provide a reference to which the input and output data can be 
normalized in a mathematical sense. 

The LCI stage involves collecting data concerning resource usage, energy and materials 
consumption, emissions and products resulting from each activity in the production system. 
As mentioned above, all these in- and out flows are calculated on the basis of the functional 
unit. In the third phase, the LCIA phase, the data collected is classified into specific 
categories and aggregated. This stage is composed of several mandatory elements and there 
are also optional elements for normalization, grouping or weighting of the indicator results 
and data quality analysis techniques. Finally, the life cycle interpretation is a procedure to 
identify, qualify, check and evaluate the information from the results of the LCI and/or 
LCIA of a product system. It is important to appreciate the reversible nature of an LCA 
study. It may be necessary, at some point, to go back to the previous stage to question and 
probe the results obtained. This is commonly done in LCA analysis, and arrows have been 
turned round to emphasize this point. 

In this study, LCA was conducted following the guidelines stated above to determine the 
potential environmental impacts of producing 1 kg of chocolate in Ghana. The boundaries of 
the system studied have been shown in Figure 1, the process flow chart for chocolate 
production. (Those processes with broken boundaries were excluded from the analysis.) The 
inputs and outputs data collected in this work from the field and standard LCA databases 
are summarized in Table 5. Using the ISO series and CML 2001 database from the Centre for 
Environmental Science at the University of Leiden for impact assessment, the results given 
in Table 6 were obtained for the quantified impact scores for the selected relevant 
environmental impact categories (Ntiamoah and Afrane, 2009). Data storage and analysis 
were performed using the GaBi 4 LCA software. 

The overall scores show that freshwater aquatic ecotoxicity and human toxicity are the most 
significant environmental impacts made by the process. In order to examine closely the 
contribution of the various stages of production to the overall impacts, Figure 3 was plotted. 
The percentage contribution of each stage to the total impact score of each category is given 
in this figure. The cocoa production stage was identified as the key life-cycle stage in terms 
of environmental impacts, as it makes the largest contribution to five out of the eight 
environmental impact categories considered in the study. The figure shows that it this is the 
most predominant contributor to eutrophication, ozone depletion, freshwater aquatic 
ecotoxicity, human toxicity, and terrestric eco-toxicity, with average contributions greater 
than 95%. Indeed the production and use of fertilizers and pesticides account for almost all 
the environmental burdens in the cocoa production stage. The significance of each of these 
environmental categories which are prominent, in cocoa production will be examined in 
turn. 



Use of Pesticides in the Cocoa Industry and Their Impact on the Environment and the Food Chain 63 



INPUTS/OUTPUTS 

Energy Inputs 

Electricity, (from national grid) 
Diesel 
Petrol 

Materials Inputs 
Water 

Fertilizer (N:P:K 0: 22:18 + 9CaO + 7S + 6MgO) 
Pesticides 

-Fungicides 

-Insecticides 
Land use 

Products/By-Products 
Chocolate 
Cocoa Liquor 
Cocoa Butter 
Cocoa Cake 
Cocoa Powder 
Cocoa Shells 

Air Emissions 
Dust (PM2,5 - PM10) [Particles to air] 
Sulphur dioxide [Inorganic emissions to air] 
Heavy metals to air 

Carbon dioxide [Inorganic emissions to air] 
Carbon monoxide [Inorganic emissions to air] 
Pesticides to air 

Water Emissions 
Biological oxygen demand (BOD) 
Chemical oxygen demand (COD) 
Nitrates 
Oil & Grease 
Phosphates 
Total dissolved solids 
Total suspended solids 
Heavy metals to fresh water 
Pesticides to fresh water] 

Soil Emissions 
Pesticides to soil 
Heavy metals to agricultural soil 



Amount 


Un 


3.1716E-01 


MJ 


5.3142E-02 


Kg 


8.9967E-03 


Kg 


5.1274E+00 


Kg 


1.4590E-01 


Kg 


7.4200E-03 


Kg 


8.0000E-04 


Kg 


3.9218E-05 


Ha 


1.0000E+00 


Kg 


3.1948E-01 


Kg 


2.3125E-01 


Kg 


2.6875E-01 


Kg 


7.5000E-02 


Kg 


9.8000E-02 


Kg 


2.5000E-03 


Kg 


8.0000E-03 


Kg 


3.5745E-05 


Kg 


2.3790E-01 


Kg 


8.4100E-03 


Kg 


8.1308E-04 


Kg 


5.0437E-12 


Kg 


9.8212E-12 


Kg 


3.7500E-15 


Kg 


1.0000E-14 


Kg 


4.4204E-14 


Kg 


5.1525E-12 


Kg 


4.1287E-12 


Kg 


7.4761E-04 


Kg 


3.6880E-03 


Kg 


9.4477E-04 


Kg 


4.1870E-05 


Kg 



(Source: Ntiamoah and Afrane, 2009.) 

Table 5. Summary of input/ output data for the production of 1 kg chocolate from Ghanaian 
cocoa beans, 2004/2005 season. 



64 


Pesticides in the Modern World - Risks and Benefits 




Environmental Impact Category 


Overall 
Impact Score 



Acidification Potential (AP) 9.7351E-03 kg S0 2 

Eutrophication Potential (EP) 9.1568E-04 kg PO4 3 - 

Freshwater Aquatic Ecotoxicity Potential (FAETP) 5.0797E+00 kg *DCB 

Global Warming Potential (GWP) 3.5602E-01 kg C0 2 

Human Toxicity Potential (HTP) 4.4426E+00 kg *DCB 

Ozone Layer Depletion Potential (ODP) 4.9805E-09 kg *R11 

Photochem. Ozone Creation Potential (POCP) 9.3002E-04 kg Ethene 

Terrestrial Ecotoxicity Potential (TETP) 6.3796E-03 kg *DCB 

*DCB is 1, 4 dichlorobenzene, *R11 is trichlorofluoromethane. 

Table 6. Characterization results (overall impact scores) for the production of 1 kg chocolate 
in Ghana, obtained by using the CML 2001 method 

Eutrophication 

Eutrophication or nutrification is a measure of the over-fertilisation of soils and 
contamination of water-bodies with nutrients. In waters it causes excessive algal growth and 
negative modification of the aquatic ecosystems resulting in oxygen depletion and death of 
certain species. In soils, on the other hand, it promotes monocultures and loss of biodiversity 
(Heijungs et al (1992) and Guinee et al (2001)). Since nitrogen and phosphorus are the 
limiting nutrients for most of the aquatic systems, leaching of these nutrients into water- 
bodies results in eutrophication. High nitrate levels have been found in drinking water in 
developing countries. This has been linked to a disease known as methaemoglobinaemia, 
commonly referred to as the blue-baby syndrome, in agricultural areas (Pretty and Conway, 
1988; Conway and Pretty, 1988). Although incidence of this disease in Ghana has not been 
reported in the literature, to the best of our knowledge, given the large amounts of fertilizer 
being used in cocoa production, possible contamination of water bodies need to be a matter 
of concern to stakeholders in the industry. 

Freshwater Aquatic, Terrestial and Human Toxicity 

From the results of Figure 2, not only are freshwater aquatic and human toxicity limited 
almost exclusively to the cocoa production stage, but they have the highest numerical values 
in the figure, which makes them more significant than the others. Terrestial toxicity, though 
not of the same magnitude as the other two, is nevertheless important. Toxicity to humans, 
flora and fauna is caused by a variety of substances, ranging from carcinogens to persistent 
toxins such as heavy metals which find their way into the food chain. The probability exists 
for harmful chemicals directly or indirectly poisoning some organisms and ultimately 
eliminating them from the ecosystem, and thereby restricting the biodiversity of the region 
and upsetting the food chain. 

Acidification 

Acidification is an indication of the gradual degradation of the soil and it is caused by acid 
solution formed when pollutants generated from the combustion of fuels are released into 
the atmosphere. In technical terms, it is caused by the build-up of protons in soils and lakes. 
Hauschild and Wenzel, (1998) describe it as a fall in the capacity of the soil to neutralize the 
acids that run through it. Higher acidity in certain types of soils can lead to the mobilisation 
of different fixed ions, which are then absorbed by plants to their detriment. Water which 



Use of Pesticides in the Cocoa Industry and Their Impact on the Environment and the Food Chain 65 



seep through acidic soils can harm aquatic ecosystems in the different lakes and rivers and 
in some severe cases, acidic water has been known to leave some water-bodies lifeless 
(Mannion and Bowlby, 1992). Acidification can be caused directly by acids and indirectly by 
acidic anhydrides (sulphur dioxide and trioxide and oxides of nitrogen) and ammonia. 



B CML2001, Photochem. Ozone Creation Potential (POCP) [kg Bhene-Equiv.] 

B CML2001, Ozone Layer Depletion Potential (ODP, steady state) [kg R11-Equiv.[ 

I I CML2001, Human Toxicity Potential (HTP inf.) [kg DCB-Equiv.] 

I I CML2001, Global Warming Potential (GWP 100 years) [kg C02-Equiv.] 

I I CM.2001, Freshw ater Aquatic Ecotoxicity Pot. (FAETPinf.) [kg DCB-Equiv.] 

I I CML2001, Eutrophication Potential (EP) [kg Phosphate-Equiv.] 

I I CML2001, Acidification Potential (AP) [kg S02-Equiv.[ 



100-j 

95- 

90- 

85- 

80- 

75- 

70- 

65- 

s 6&j 

I 55- 

* 50J 

| 45J 

°> 4&| 

35- 

30- 

25- 

20- 

15- 

10- 

5- 

0- 



w 



JL 



a 



n 




n 



n 



Chocolate manufacturing 



Cocoa processing 



Cocoa production 



Fig. 3. Relative contribution of different stages of the life-cycle to the various environmental 
categories 

Ozone layer Depletion Potential 

The thinning of the ozone layer in the stratosphere is allowing increased levels of ultraviolet 
radiation to reach the earth, leading to diseases in humans (skin cancer and cataracts) and 
adverse effect on ecosystems. Ozone layer depletion is caused by the emission of halons and 
CFCs during the production of pesticides. These processes are based on complicated 
reaction systems, including both solid phase and gaseous phase reactions, and a limited 
number of substances are involved (Hauschild and Wenzel, 1998). Most notably methane, 
nitrous oxide, water vapour, chlorine and some bromine compounds, are responsible for the 
breakdown of ozone molecules. Human activities have increased the amount of substances 
involved in the breakdown of ozone and especially stable, long-lived chlorine and bromine 
containing hydrocarbons (i.e. chlorofluorocarbons or CFCs, tetrachloromethane, 
trichloroethane, etc.) are believed to contribute significantly. Fortunately the contribution to 
ozone-layer thinning, as a result of cocoa production and processing, turns out to be the 
least significant, according to Figure 3. 



6. Conclusion 

The use of pesticides is often advisable and sometimes essential when a crop is threatened. 
Integrated pest management is a concept which is now generally known and widely 



66 Pesticides in the Modern World - Risks and Benefits 

accepted, and it is hoped that the judicious use of pesticides will be accepted as an integral 
part of pest management strategy. Technologies are presently available for the safe use of 
pesticides in cocoa and awareness of their correct and proper use needs to be stimulated 
(Bateman, 2008). However, introducing Good Agricultural Practices to the more than three 
million (often illiterate) smallholder farmers in the world cocoa economy is a major 
challenge. Ghana is making some strides in this area. 

The clear indications are that the current agricultural practices for cocoa production are not 
sustainable, from both the environmental and economic perspective. Continued increase in 
the costs and amounts of chemicals put into the environment does not portend well for the 
future of this cash crop. The study has shown that current pests and diseases control 
practices in Ghanaian cocoa production which rely primarily on chemical methods, though 
well administered, results in more environmental damage. In the long term integrated pest 
management (IPM), which encourages natural control of pest populations, promises to 
reduce the use of pesticides. Some of the techniques used in this approach include 
enhancing natural enemies, planting pest-resistant crops, and, when absolutely necessary, 
efficient and judicious use of pesticides. 

Pesticides continue to be attractive to most farmers and governments because they are 
simple to use, compared to the IPM methods, and returns on investments are not only good, 
but are predictable. A switch to IPM must be preceded by careful planning, and intensive 
education and training at the farm level, along with continuing research. In addition, 
promoting IPM will definitely require adjusting those subsidies and policies that encourage 
extensive pesticide use; otherwise farmers may not be able to resist the temptation of going 
back to their old ways. 

7. Acknowlegements 

The readiness of the staff of CODAPEC, Research and HI-TECH Divisions of COCOBOD, 
Accra, and the Cocoa Research Institute of Ghana at Tafo, to assist during the data collection 
stage was commendable and is hereby acknowledged. 

8. References 

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1943;55(15). 
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Conway, G. R. and Pretty, J. N. 1988, 'Fertilizer risks in developing countries', Nature, Vol. 

334, pp. 207-208. 
Drolet R, Arendt TD, Stowe CM. (1984) Cacao bean shell poisoning in a dog. JAVMA 1984; 

185(8): 902. 
Entwistle, P. F. (1972): Pests of Cocoa. Longman Tropical Science Series, London, UK 
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Operational Guide to the ISO Standards. Kluwer Academic Publishers. The 

Netherlands. 
Hauschild, M., Wenzel, H (1998). Environmental assessment of products. Volume 2: 

Scientific background. London, UK, Chapman & Hall. 
Heijungs, R. (1992) (editor): Environmental Life Cycle Assessment of Products - Guide. 

Center for Environmental Science, University of Leiden, The Netherlands. 
ICCO, 2010: ICCO Quarterly Bulletin of Cocoa Statistics, Vol. XXXVI, No. 4, Cocoa year 

2009/2010. Published: 30-11-2010 

(www icco .org) 
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133 rd Meeting, EBRD Offices, London, 5-7 June, 2007 
ISO 14040 (1997): Environmental management - Life cycle assessment - Principles and 

framework. International Organization for Standardization; Technical Committee 

ISO/TC 207/ SC5. 
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definition and inventory analysis. International Organization for Standardization; 

Technical Committee ISO/TC 207/SC5. 
ISO 14042 (2000a): Environmental management - Life cycle assessment - Life cycle impact 

assessment. International Organization for Standardization; Technical Committee 

ISO/TC 207/ SC5. 
ISO 14043 (2000b): Environmental management - Life cycle assessment - Life cycle 

interpretation. International Organization for Standardization; Technical 

Committee ISO/TC 207/SC5. 
Johnson, E. S.; Aime, M.C.; Crozier, J.; Flood J.; Iwaro, DA.; and Schnell, R.J.: A new 

morpho-fype of Phytophthora palmivora on cacao in Central America, under IPM- 

Advances in Conventional Methods, in Proceedings of the 5 th INCOPED 

International Seminar on Cocoa Pests and Diseases, Akrofi, A.Y., Baah, F.(Eds) 

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68 Pesticides in the Modern World - Risks and Benefits 

Mahot, H.; Babin, R.; Dibog, L.; Tondje, P.R.; and Bilong, C. (2005): Biocontrol of cocoa mirid 

Sahlbergella singularis hagl. (Hemiptera: Miridae) with Beauveria bassiana 

Vuillemin: First results of activities carried out at IRAD, Cameroon in Proceedings 

of the 5 th INCOPED International Seminar on Cocoa Pests and Diseases, Akrofi, 

A.Y., Baah, F.(Eds) INCOPED Secretariat, Cocoa Research Institute, Tafo, Ghana 
Moy, G.G.; Wessel, J.R. (2000): Codex Standard for Pesticides Residues, in Internatioanl 

Standards for Food Safety, Rees, N.; Watson, D., (Eds) Aspen Publishers Inc. 

Gaithersburg, MD, USA 
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Chichester 
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Yanful, Ernest K. (Editor) (2009), Springer. ISBN: 978-1- 40209138-4. Pp.35 - 41. 
Offei, S.K.; Cornelius, E.W.; Sakyi-Dawson, 0.(2008): Crop Diseases in Ghana and their 

Management, Sponsored by TALIF for College of Agriculture, University of Ghana, 

Legon, Accra, Smartline Ltd, ISBN 9988600275 
Opoku, I.Y., Akrofi, A.Y., Appiah, A.A.(2007): Assessment of sanitation and fungicide 

application directed at cocoa tree trunks for the control of Phytophthora black pod 

infections in pods growing in the canopy, Eur J Plant Pathol (2007) 117:167-175 
Opoku, I.Y., Gyasi, E. K., Onyinah, G K., Opoku, E., Fofie, T.: The National Cocoa Diseases 

and Pests Control (CODAPEC) 
Owusu-Manu, E. (1977) Insecticide residues and tainting in cocoa, Pesticide Management and 

Insecticide Resistance, 1977, Academic Press Inc, London 
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Diseases and Pests Control(CODAPEC) Programme: Achievements and 

Challenges. Proceedings of the 15 lh International Cocoa Research Conference. San 

Jose, Costa Rica. 9 - 14* October, 2006. Pp. 1007 - 1013. 
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4 October - December 2008. ISSN 

0855-7918. 
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High Risk in the Tropics? International Institute for Environment and 

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contamination in cocoa and cocoa products: isotopic evidence of global contamination. 

Environmental Health Perspectives 113 (10): 1344-1348. 
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Feb. 19, 2007 www.dropdata.org/cocoa/index.htm (The World's Worst Cocoa 

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effect on health, March 2011) 
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1991/1992, Third Edition, Nairobi, Kenya. 



Industrial Contaminants and 
Pesticides in Food Products 

Ruud Peters 1 , Henry Beeltje 2 and Marc Houtzager 2 

^IKILT - Institute of Food Safety, 
2 TNO Built Environment and Geosciences, Environment, Health and Safety, 

Netherlands 



1. Introduction 

In our modern world a large number of man-made chemicals are being used. As a 
consequence their widespread presence in the environment is becoming increasingly well 
documented (Vethaak et al, 2002; Peters et al., 2008). They are found in a vast range of 
consumer products and include plasticizers, emulsifiers, flame retardants, perfluorinated 
compounds, artificial musks and organotin compounds. While they have undoubtedly 
improved the quality of our lives, a consequence of their intensive use is a widespread 
presence in the environment. Human exposure to these compounds may be through contact 
with consumer products containing such chemicals as additives, but also through food 
products. Since many of these compounds have a lipophilic nature there is a potential for 
bio-accumulation through the food chain especially in products with a high fat content. This 
is reflected in the presence of persistent organic compounds such as organochlorine 
pesticides and polychlorinated biphenyls that can be found in food products although there 
use has been seized many years ago. Many of these compounds have also been found in 
human blood indicating that humans are exposed to these chemicals (CDC, 2001, 2003; 
Guenther et al., 2002). This exposure may be through different routes. One is the use of these 
chemicals as additives in consumer products such as carpets, curtains, toys and electronic 
equipment. The exposure of these chemicals in house dust indicates the potential for human 
exposure. Another route for human exposure is, of course, through food products. Since 
many of these compounds have a lipophilic nature, they can be bio-accumulated through 
the food chain especially in products with a high fat content. This study focused on the 
presence and concentrations of a number of typical man-made chemicals in food products 
that many of use daily. The chemicals considered in this study are: brominated flame 
retardants (BFR's), phthalates, artificial musks, alkylphenols (AP's), organochlorine 
pesticides (OCP's), polychlorobiphenyls (PCB's), organotin compounds (OT's) and 
perfluorinated compounds (PFC's). 

2. Methods and materials 

2.1 Sampling and sample pre-treatment 

All samples, mostly fresh food products were purchased in regular shops in nine European 
countries including the Netherlands, the United Kingdom, Germany, Finland, Sweden, 



70 Pesticides in the Modern World - Risks and Benefits 

Spain, Poland, Italy, Estonia and Greece. Samples were sent to the laboratory where 
laboratory samples were prepared and stored at -18°C until analysis. In general, solid food 
samples were cut into small pieces and homogenised with a blender. If not the entire sample 
was used or homogenised, proportional sub-sampling was applied and the collected sub- 
samples were homogenised. Milk was acidified with formic acid and the solid part 
containing the proteins and fat was separated from the liquid phase. Both parts were stored 
for analysis. Orange juice was centrifuged and vacuum filtrated and the solid and liquid 
parts were stored for analysis. A selected number of chemical parameters were determined 
in each sample, based on expectations and reports in the literature. 

2.2 Chemical parameters 

The chemical parameters determined in this study are listed in table 1, including the 
abbreviations that are used throughout the text and in the result tables. Note that not all 
parameters are determined in all samples. 

2.3 Analytical procedures 

For the determination of the OCP's, PCB's, BFR's, phthalates and artificial musks, a weight 
sub-sample of the homogenised laboratory sample was mixed with anhydrous sodium 
sulphate in a mortar and spiked with internal standards. The internal standards used were 
13 C-labelled standards for PCB's and BFR's, 2 D-labelled standards for OCP's and phthalates, 
and a surrogate standard for the artificial musks. The samples were Soxhlet extracted for 16 
hours using a mixture of 10% diethyl ether in hexane. For milk and orange juice a 
proportional amount of the liquid phase was pre-extracted with hexane and this hexane 
extract was used in the Soxhlet extraction of the solid part of these samples. Olive oil was 
directly diluted in hexane. One procedural blank, consisting of 40 g anhydrous sodium 
sulphate, was included in every batch of 10 samples. All extracts were concentrated to a 
volume of 50 ml and split into two equal parts of 25 ml. For the determination of the OCP's, 
PCB's and BFR's, one part of the extract was washed several times with sulphuric acid of 
increasing concentration to remove the major part of the lipids. The remaining extract was 
concentrated and purified over a glass chromatographic column packed with florisil and 
capped with anhydrous sodium sulphate to isolate the fraction containing the OCP's, PCB's, 
PBDE's and HBCD. The eluent was concentrated to a small volume and a syringe standard 
(1,2,3,4-tetrachloronaphthalene) was added. This final extract was analysed on an Agilent 
6890 series gas chromatograph coupled to an Agilent 5973 mass spectrometer (GC/MS) and 
equipped with a HP-5-MS, 30 m x 0.25 mm (i.d.), film thickness 0.25 jim, fused silica 
capillary column. The mass spectrometer was operated in the selected ion monitoring mode 
and typically two or three characteristic ion masses were monitored for each analyte. The 
samples were analyzed for the following OCP's; a-, p- and y-hexachlorohexane (HCH), 
hexachlorobenzene (HCB), a- and p-chlordane, o,p'-, p,p'-DDE, o,p'-, p,p'-DDD and o,p'-, 
p,p'-DDT: The following PCB congeners: 18, 28/31, 22, 41/64, 44, 49, 52, 54, 56/60, 70, 74, 
87, 90/101, 99, 104, 105, 110, 114, 118, 123, 138, 141, 149, 151, 153/168, 156, 157, 158, 167, 170, 
177, 180, 183, 187, 188, 189, 194, 199 and 203: The following PBDE congeners: 17, 28, 32, 35, 
37, 47, 49/71, 66, 75, 77, 85, 99, 100, 119, 126, 138, 153, 154, 156, 166, 181, 183, 184, 190, 191, 
196, 197, 206, 207 and 209 and HBCD. 

For the determination of the phthalates and artificial musks the second part of the extract 
was purified using a dimethylformamide-hexane partitioning to remove lipids. In this 



Industrial Contaminants and Pesticides in Food Products 



71 



Organochlorine pesticides (OCPs): 


Phthalates: 


a-hexachlorohexane (a-HCH) 


di-methyl phthalate (DMP) 


(?-hexachlorohexane (p-HCH) 


di-ethyl phthalate (DEP) 


y-hexachlorohexane (y-HCH) = lindane 


di-isobutyl phthalate (DiBP) 


hexachlorobenzene (HCB) 


di-butyl phthalate (DBP) 


a-chlordane 


benzylbutyl phthalate (BBP) 


(?-chlordane 


di-(2-ethylhexyl) phthalate (DEHP) 


o,p'-DDE 


di-isononyl phthalate (DiNP) 


p,p'-DDE 


di-isodecyl phthalate (DiDP) 


o,p'-DDD 




p,p'-DDD 




o,p'-DDT 




p,p'-DDT 





Polychlorinated biphenyls (PCBs): 


Polybrominated diphenylethers (PBDEs): 


PCB-18 


PCB-118 


BDE-17 


BDE-138 


PCB-28/31 


PCB-123 


BDE-28 


BDE-153 


PCB-22 


PCB-138/158 


BDE-32 


BDE-154 


PCB-41 


PCB-141 


BDE-35 


BDE-156 


PCB-44 


PCB-149 


BDE-37 


BDE-166 


PCB-49 


PCB-151 


BDE-47 


BDE-181 


PCB-52 


PCB-153/168 


BDE-49/71 


BDE-183 


PCB-54 


PCB-156 


BDE-66 


BDE-184 


PCB-56/60 


PCB-157 


BDE-75 


BDE-190 


PCB-64 


PCB-167 


BDE-77 


BDE-191 


PCB-70 


PCB-170 


BDE-85 


BDE-196 


PCB-74 


PCB-177 


BDE-99 


BDE-197 


PCB-87 


PCB-180 


BDE-100 


BDE-206 


PCB-90 


PCB-183 


BDE-119 


BDE-207 


PCB-99 


PCB-187 


BDE-126 


BDE-209 


PCB-101 


PCB-188 






PCB-104 


PCB-189 


Other brominated flame retardents: 


PCB-105 


PCB-194 




PCB-110 


PCB-199 


tetrabromobisphenol-A (TBBPA) 


PCB-114 


PCB-203 


hexabromocyclododecane (HBCD) 



Perfluorinated chemicals (PFCs): 


Artificial musks: 


perfluoro-octanoic acid (PFOA) 


galaxolide (HHCB) 


perfluoro-octane sulphonate (PFOS) 


tonalide (AHTN) 


perfluoro-octane sulfonamide (PFOSA) 


musk xylene (MX) 


perfluoro-nonanoic acid (PFNA) 


musk ketone (MK) 


perfluoro-decanoic acid (PFDA) 




perfluoro-undecanoic acid (PFUnA) 




perfluoro-dodecanoic acid (PFDoA) 


Organotin compounds: 


perfluoro-tetradecanoic acid (PFTrA) 






mono-butyltin (MBT) 


Alkylphenols (AP): 


di-butyltin (DBT) 




tri-butyltin (TBT) 


nonylphenol isomers (NP) 


mono-octyltin (MOT) 


octylphenol isomers (OP) 


di-octyltin (DOT) 



Table 1. Chemical parameters determined in this study including abbreviations used in the 
text and result tables 



72 Pesticides in the Modern World - Risks and Benefits 

partitioning the hexane extract is extracted with dimethylformamide (DMF) to isolate the 
phthalates and artificial musks. After removal of the hexane layer, water is added to DMF 
fraction and the analytes are re-extracted into fresh hexane. This extract was concentrated 
and purified over a glass chromatographic column packed with florisil and capped with 
anhydrous sodium sulphate to isolate the fraction containing the phthalates and artificial 
musks. The eluent was concentrated to a small volume, the syringe standard was added and 
this final extract was analysed with the identical GC/MS system as described above. The 
mass spectrometer was operated in the selected ion monitoring mode and typically two or 
three characteristic ion masses were monitored for each analyte. The samples were analyzed 
for the following phthalates; di-methyl- (DMP), di-ethyl- (DEP), di-isobutyl- (DIBP), di- 
butyl- (DBP), benzylbutyl- (BBP), di-(2-ethylhexyl)- (DEHP), di-isononyl- (DINP) and di- 
isodecyl phthalate (DIDP): The following artificial musks; musk ketone (MK), musk xylene 
(MX), tonalide (AHTN) and galaxolide (HHCB). 

AP's and the brominated flame retardant tetrabromobisphenol-A (TBBPA) were isolated 
using a steam distillation procedure described by Guenther et al., 2002. In the round bottom 
flask of the steam distillation apparatus typically 10 g of the laboratory sample was spiked 
with an internal standard and mixed with 250 ml Milli-Q water to which was added 1 ml of 
concentrated hydrochloric acid and 20 g of sodium chloride. During the overnight 
distillation process the organic phenols are isolated in the organic solvent, in this case 
hexane, in the backflow cooling system of the apparatus. The hexane extract is isolated, 
dried with anhydrous sodium sulphate and concentrated. Following drying the extract was 
reduced in volume nearly to dryness under a stream of nitrogen and re-dissolved in 1 ml of 
methanol. During concentration of the hexane extract care has to be taken to avoid losses of 
the AP's and rinsing of the glass surfaces with methanol is necessary. The methanol extract 
is analysed with liquid chromatography coupled with mass spectrometry (LC/MS) in the 
selected ion monitoring mode. 

For the determination of perfluorinated compounds, specifically perfluoro-octanoate 
(PFOA), perfluoro-octane sulphonate (PFOS) and perfluoro-octane sulphonamide (PFOSA) 
sub-samples of 5 gram were collected in 50 ml poly-propylene tubes and extracted using 
acetonitrile. The samples were centrifuged and the clear liquid is decanted and purified over 
a glass chromatographic column packed with florisil, silica, LC-NH2 and activated carbon. 
The residue in the poly-propylene tube is extracted two times more and each extract is 
decanted over the same chromatographic column, thus combining the purified extracts. 0.5 
ml octanol is added as a keeper and the extracts are concentrated to a small volume. 

3. Results 

3.1 Validation of analytical methods 

The analytical methods used were validated previous to the execution of this study. The 
parameters determined were linearity, repeatability, recovery from the rainwater matrix and 
method detection limits (MDL). For the determination of the organochlorine pesticides, 
polychlorinated biphenyls, brominated flame retardants, phthalates, artificial musks and 
organotin compounds internal standards were added to each sub-sample prior to analysis. 
For the perfluorinated compounds two relevant samples are spiked with the compounds of 
interest and analysed. The recovery of the internal standard and spikes were in the range of 
70 to 140% with the exception of artificial musks where recoveries in the range of 56 to 87% 
were found, and the organotin compounds where recoveries were in the range of 67 to 99%. 



Industrial Contaminants and Pesticides in Food Products 



73 



With the exception of PCBs and organotin compounds, the results are not corrected for the 
recovery of the internal standards or spikes since the spikes used were not compound 
specific and their recovery is only used to evaluate the performance of the method. With 
each series of ten samples a blank sample was included. For the blank analysis the complete 
analytical procedure was followed, including all chemicals and solvents, but no sample was 
added. Blank results were only found for the phthalates DEHP, DIBP and DBP, The results 
were corrected for these blank values and the detection limits were raised to 10 ng/g for 
DIBP and DBP, and to 20 ng/g for DEHP. 



3.2 Organochlorine pesticides 

Organochlorine pesticides (OCPs) include compounds like DDT, lindane, 
hexachlorobenzene and chlordane, among others. DDT is a well-known agricultural 
insecticide that has been used extensively on a global basis for over 40 years. Although their 
manufacture and application are now largely prohibited or restricted in industrialized 
western countries because of their toxicity and persistence, they can still be found in 
environmental and biological matrices due to their persistence. Pesticide exposure has been 
associated with arthritis, diabetes, neurobehavioral changes and DNA damage Cox et al., 
2007; Lee et al. 2007; Jurewicz et al., 2008; Rusiecki et al., 2008). The structures of p,p'-DDT 
and it's breakdown product p,p'-DDE, are shown below. 





p,p '-DDT PP '- DDE 

As a part of its monitoring program, the Food and Drug Administration (FDA) determines the 
levels of pesticide residues in a wide variety of foods typically consumed by Americans. Over 
the past ten years, these surveys have detected DDE and other OCPs in a variety of foods 
including meat, fish and shell fish products, eggs, root vegetables, legumes (beans, peas, and 
peanuts), some fruits, and leafy greens. In 1999 DDE or DDT were detected in 22% of the 1,040 
food items analysed in the FDA Total Diet Study (FDA, 1999). The results for the 2003 Total 
Diet Study indicate DDT, but mainly DDE in 18% of the various food items in concentrations 
ranging from 0.1 to 11 ng/g (FDA, 2003). Those for chlordane and lindane range from 0.1 to 3.8 
and from 0.1 to 8.4 ng/g product. In general, the concentrations as well as the frequency of 
detection of OCPs were lower in the 2003 study. The results of the OCPs in this study are 
presented in table 2 at the end of this section, and graphically in figure 1. OCPs are found in 17 
of the 25 samples. The predominant OCPs that are detected are p,p'-DDE and HCB both found 
in 15 of the 25 samples. In addition o,p'-DDE and cis-chlordane were found in one and two 
samples respectively. The maximum concentration found for p,p'-DDE was 5.6 ng/g in a 
sample of pickled herring. The median concentrations for p,p'-DDE and HCB were 0.43 and 
0.14 ng/g. Compared to the FDA's Total Diet Study, p,p'-DDE and HCB are found more 
frequently but in lower concentrations. This compares with a recent study of Schecter et al. 
who reported p,p'-DDE in 23 out of 31 samples in concentrations ranging from 0.06 to 9.0 ng/ g 
with a median value of 0.51 ng/g (Schecter et al., 2010). 



74 



Pesticides in the Modern World - Risks and Benefits 





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n 




m g a | u 
(/I 


Milk 

Olive oil 

Chicken 

Fish fingers 

Salmon 

Honey 

Orangejuice 

Tuna 

Brown bread 

Frankfurter sausages 

Reindeer meat 

Pickled herring 

Minced beef 

Pork chops 

Cottage cheese 

Salami 

Cheese 

Ham 

Cheese 

Scottish cheese 



Fig. 1. Graphic presentation of the total-OCP concentration in 25 food items 

3.3 Polychlorinated biphenyls 

Polychlorinated biphenyls (PCBs) are marketed as cooling or insulating fluids for 
transformers, as softeners in the varnish and adhesive industries, and as hydraulic fluids. 
These compounds are not combustible, are heat resistant and make good solvents. On the 
other hand there is a severe toxic effect of PCBs, which damage the organs responsible for 
metabolism and also the nervous system (Guo et al., 1995; Ribas-Fito et al., 2001). Because of 
their persistence, PCBs are widely spread in the environment and, due to their good lipo- 
solubility, are easily deposited and concentrated in human, animal and plant tissue. The 
structures of two of the seven indicator PCBs, PCB-28 and PCB-180 are shown below. 




2,4 l 4 l -trichlorobiphenyl (PCB 28) 2,2' ,3,4,4' ,5,5'-heptacrilorobiprienyl (PCB 180) 

In the Total Diet Study of 2003 the levels of the sum of i-PCBs (e.g. PCB-28, PCB-52, PCB- 
101, PCB-118, PCB-138, PCB-153 and PCB-180) ranged from 6 to 70 ng/g product. The 
Estonian Environment Research Centre determined levels of the sum of i-PCBs in Estonian 
butter and found a relative narrow concentration range of 5.2 to 8.8 ng/g lipid, translating 
to a range of 4.2 to 7.0 ng/g on a wet weight basis (EU, 2004). A study of the European Food 
Safety Association (EFSA) of different food types sampled across Europe between 1997 and 
2003 reports median concentrations for the sum of i-PCBs ranging from 1.4 to 12 ng/g. Only 
fish oil showed a higher median concentration of 52 ng/ g for the sum of i-PCBs (Gallani et 
al., 2004). The results for the PCBs in this study are presented in table 2 at the end of this 



Industrial Contaminants and Pesticides in Food Products 75 

section. A graphic summary is given in figure 2. PCBs are found in every sample with PCB- 
18, -28 and -52, with the highest frequency. The highest PCB concentrations were found in 
the pickled herring and the salmon with concentrations for the sum of i-PCBs of 13 and 3.3 
ng/g. The highest concentration of an individual PCB was for PCB-153, 5.5 ng/g in the 
salmon. The results found are comparable with those reported by EFSA for different food 
types sampled across Europe. 



"3 7 - 

& 6 - 

5 5 ~ 

8 3 - , 



1 I _ ■ - B H 



n.l.H.B.B. B .H.B 



5 o 



Fig. 2. Graphic presentation of the total-PCB concentration in 24 food items 

3.4 Brominated flame retardants 

Brominated flame retardants (BFRs) are widely used in electronic household equipment, 
plastics, textile and polyurethane foam in furniture and cars for safety reasons. Of the 
brominated products, about one-third contain tetrabromobisphenol-A (TBBPA) and 
derivatives, another third contains various bromines, including hexabromocyclododecane 
(HBCD) and the last third contains polybrominated diphenylethers (PBDEs). All three types 
of BFRs are determined in this study. The PBDEs are commercial mixtures with different 
degrees of bromination and used as additives to fireproof polymers. HBCD is a cyclo- 
aliphatic brominated chemical introduced as a replacement for the PBDEs and with the 
same applications. TBBPA is mainly used as a reactive (chemically bound) flame retardant 
in epoxy polymers such as printed circuit boards in electronic equipment. The structure of 
BDE-209 (better known as deca-BDE), HBCD and TBBPA is shown below. In a study by the 
Dutch National Institute for Public Health and the Environment the levels of various PBDEs, 
HBCD and TBBPA in 84 food products were determined (de Winter-Sorkina et al., 2003, 
2006). With the exception of fish products PBDEs were absent or present in low 
concentrations (<0.1 ng/g) in food products. For the fish products the concentrations of the 
congeners BDE-28, -47, -99, -100, -153 and -154 ranged from 0.1 to 14 ng/g. BDE-209 was 
found in none of the 84 products while TBBPA was found in 7 products in concentrations 
ranging from 0.1 to 3.4 ng/g. Surprisingly, HBCD was found in 28 of the 84 samples in 
concentrations ranging from 0.1 to 8.9 ng/g product. TBBPA and HBCD were 
predominantly found in fish products, especially in eel. 



76 



Pesticides in the Modern World - Risks and Benefits 





tetrabromo bisphenol-A (TBBA) 



Br Br 

decabromo diphenylether (BDE 209) 
A recent report on the results of a round robin exercise for BFRs in environmental, human 
and food samples gives some results for herring and salmon. The highest concentrations are 
found for BDE-47, up to 9.3 ng/g in herring and 0.89 ng/g in salmon. Those for the BDE-28, 
-99, -100, -153, -154 and -183 are in the range of 0.1 to 1.3 ng/g. BDE-209 and HBCD were not 
detected in these samples. The results for PBDEs in typical market basket studies show some 
differences, while studies in Spain (Bocio et al., 2003) and Japan (Ohta et al., 2002, 2008) 
show a predominance of the tetra- and penta-BDE with maximum concentrations up to 0.34 
ng/g, an American study reports PBDE levels up to 3.1 ng/g product with surprisingly a 
predominance of BDE-209 which comprised as much as 50% of the total PBDE content in 
some of the samples (Schecter et al., 2004). The results for the BFRs in this study are reported 
in table 3 at the end of this section, and graphically in figure 3. PBDEs were found in 19 of 
the 24 samples. BDE-209, TBBPA and HBCD were found in none of the samples. BDE-47, -32 
and -99 seem to be the predominant and the highest concentration for an individual PBDE 
was 0.65 ng/g found for BDE-47 in the sample of Scottish Cheese. Surprisingly, and 
different from other studies, only a limited number of BFRs were found in the fish products, 
in salmon no BFRs were found at all. The total PBDE concentrations ranged from 0.15 to 
1.2 ng/g with the highest concentration in the sample of minced beef. The concentrations 
found in this study are therefore comparable with those found in the Spanish and Japanese 
study and much lower than those found in the American food study. 




Fig. 3. Graphic presentation of the total-BFRs concentration in 24 food samples 



Industrial Contaminants and Pesticides in Food Products 77 

3.5 Perfluorinated compounds 

Perfluorinated compounds (PFCs) are synthetic compounds characterised by an alkyl chain 
in which the hydrogen atoms are completely replaced by fluorine atoms. PFCs are heat 
stable, very resistant to degradation and environmental breakdown and have an 
amphiphilic nature (they repel water as well as oil). Because of these properties PFCs are 
used a myriad of applications, such as non-stick pans, stain and water repelling coatings for 
clothing, furniture and paper with typical brand names as Teflon, Gortex, Stainmaster and 
Scotchguard (3M-company, 1999). PFCs accumulate in the environment and they have been 
detected far from manufacturing plants in birds, marine plants and mammals from the 
Arctic to the Pacific and Indian Oceans and in land creatures in Europe and the USA 
(Kannan et al., 2002; Martin et al., 2003, 2004). In addition PFCs have been found in human 
blood (Kannan et al., 2004; Peters, 2005). The structures of the two most common PFCs, 
PFOA and PFOS are given below. 





perfluoro-octanoic acid (PFOA) perfluoro- octane sulfonate (PFOS) 

Many studies focus on biota such as fish and birds and only limited information about levels 
of PFCs in food seem to be available. In 2001 the Centre Analytical Laboratory performed a 
study for the 3M-company as part of a Multi-City Study. PFCs were found in a limited 
number of samples. PFOS was found in five samples, four whole milk samples and a 
ground beef sample in concentrations up to 0.85 ng/g. PFOA was found in seven samples, 
two ground beef samples, two bread samples, two apple samples and one green been 
sample in concentrations up to 2.35 ng/g (Centre Analytical Laboratory, 2001). The results 
for PFCs are given in table 3 at the end of this section. PFOS and PFOSA are found in only 
one of the five samples that were analysed. The concentrations found in the sample of 
pickled herring, are 1.3 ng/g for PFOS and 0.2 ng/g for PFOSA. PFOS is widely detected in 
the environment, animals and humans and therefore expected. Surprisingly, Schecter et al. 
did not find PFOS, but do find PFOA in 50% of the analysed samples in concentrations 
ranging from 0.02 to 1.8 ng/g (Schecter et al., 2010). 

3.6 Phthalates 

Phthalates are one of the most ubiquitous classes of chemical contaminants in our everyday 
environment as a consequence of their high volume uses in open applications. They are 
used as plasticizers to increase the flexibility of high molecular weight polymers (mainly in 
PVC), as heat-transfer fluids and as carriers, and can be found in ink, paint, adhesives, 
pesticides, vinyl flooring (Vethaak et al., 2002), but also in cosmetics and personal care 
products. Consequently, the potential for human exposure is very high. Di-(2-ethylhexyl) 
phthalate (DEHP) and di-ethyl phthalate (DEP) are two of the most common used 
plasticizers. DEHP is nowadays gradually replaced by iso-alkyl phthalate mixtures like di- 
isononyl phthalate (DINP). The chemical structure of DEP and DEHP is shown below. 



78 



Pesticides in the Modern World - Risks and Benefits 







DEP DEHP 

There is not much information about concentrations of phthalates in food. Most attention has 
been focused on phthalates in plastic wrapping materials for food products. An older study 
dating from 1994 deals with the determination of DEHP in milk, cream, butter and cheese 
(Sharman et al., 1994). DEHP was found in all these products in concentrations ranging from 
330 to 980 ng/g. More recent information is available from the UK Food Standard Agency and 
is concerned with the presence of phthalates in infant formulae (Joint Food Safety and 
Standards Group, 1998). Seven phthalates including DEHP were determined in 39 samples of 
infant formulae. In 12 of the 39 samples none of the phthalates were found. In the remaining 
samples di-butyl phthalate (DBP), benzylbutyl phthalate (BBP) and DEHP were found. DEHP 
was the most abundant individual phthalate in concentrations ranging from 50 to 440 ng/ g 
product. DBP was found in concentrations up to 90 ng/g and BBP up to 15 ng/g product. 
Concentrations of other phthalates were less than 10 ng/g. The results for phthalates in this 
study are presented in table 3 at the end of this section, and graphically in figure 4. 16 of the 19 
samples analysed for phthalates did contain one or more of these compounds. In eggs, milk 
and orange juice no phthalates were detected. DIDP was the only phthalate that was not found 
in any of the samples. As expected DEHP is the predominant phthalate found in 16 of the 19 
samples with concentrations ranging from 20 to 24,000 ng/g. It should be mentioned that the 
latter concentration is an exception and was found in the sample of olive oil. The neck of the 
olive oil bottle contains a polymer spout that may be responsible for the high DEHP 
concentration in the olive oil. Other phthalates that are frequently found (>50% of the samples) 
are DBP and BBP, be it in lower concentrations than DEHP. 




Fig. 4. Graphic presentation of the sum of the eight phthalates in 19 food items 



Industrial Contaminants and Pesticides in Food Products 



79 



3.7 Alkylphenols 

Alkylphenols, but primarily alkylphenol ethoxylates are used as additives in plastics and as 
surface-active ingredients in industrial detergents and emulsifiers. The ethoxylates are 
produced by a condensation reaction of alkylphenols with ethylene oxide. Alkylphenols 
commonly used are nonylphenol (NP) and to a lesser extent octylphenol (OP), in both cases 
pre-dominantly the para-substituted isomers (>90%). Alkylphenols are the common 
products of bio- or chemical degradation of the ethoxylates. The chemical structure of n-NP 
is shown below. 




As with the phthalates only little information seems to be available about levels of 
alkylphenols in food. Guenther et al. determined NPs in 60 different commercially available 
foodstuffs and concluded that NPs are ubiquitous in food (Guenther et al., 2002). The 
concentrations of NPs (sum of the isomers) varied between 0.1 and 19.4 ng/g product and 
were found in all samples. Despite the lipophilic properties of NPs, high concentrations of NPs 
were not only found in fatty foods but also in non-fatty food products. In another study OP 
and NP were determined in composite foods (Fernandes et al., 2003). OP was found in only 
one sample in a concentration of 8.7 ng/g while NP was found in concentrations up to 25 
ng/g. In a previous TNO study alkylphenols were determined in wrapped fresh meat and 
cheese products (Geenen, 2003). Since the alkylphenols were determined in slices of the 
product collected directly below the foil or wrapper, the results are not representative for the 
entire product. OP was detected in none of the samples while NPs were found in five of the 
eight sub-samples in concentrations ranging from 9 to 590 ng/g. For one sample the whole 
food item was analysed resulting in a much lower concentrations in the order of 1 ng/g for 
NP. The results for alkylphenols in this study are given in table 3 at the end of this section. NP 
was found in 2 of the 19 samples, the samples of butter and bacon in concentrations around 5 
ng/g. OP was found in none of the samples. Although the concentrations are in the range of 
what Guenther found, the results are different because the frequency of detection in this study 
is far lower. Perhaps this is a result of the way sub-samples are collected since higher 
concentration may be found in top-layers beneath the packaging foil. 



3.8 Musk compounds 

In nature, musk is a compound produced by a gland in male deer which has been used in 
perfumes, but the increasing demand resulted in the production of artificial musk 
compounds. The most well-known are nitro musks like MX and MK that are nowadays 
replaced by polycyclic musks like AHTN and HHCB. Musks are used as additives for 
perfumes, in detergents and soaps, in body lotions and deodorizers. The structure of MK 
and HHCB is presented below. 



w 



V 



W 



V 



/v 

' N'^=0 




HHCB 



80 Pesticides in the Modern World - Risks and Benefits 

As far as we know there are no reports or studies concerning the presence of artificial musks 
in food or food products. However, since 1981 it is known that artificial musks can be found 
in fish (Yamagishi et al, 1981, 1983; Rimkus & Wolf, 1995; Fromme et al., 2001; Gatermann et 
al., 2002), and as a results artificial musks may be present in fish products. The results for 
musks in this study are given in table 3 at the end of this section. The nitro-musks MK and 
MX are not found in the four fish products analysed for artificial musks. The polycyclic 
musks AHTN and HHCB are found in two of the samples, the samples tuna and pickled 
herring in a maximum concentration of 0.56 ng/g for the latter sample. As in other 
environmental matrices and human blood the HHCB concentrations are about twice that of 
the AHTN concentrations. That the concentrations are lower than those reported for fish in 
the literature is probably because most literature studies report results for fish in waterways 
connected to sewer effluents and not for typical marine fish species. 

3.9 Organotin compounds 

The main OTCs to be found in food are likely to be tri-substituted compounds, tributyltin 
(TBT) and triphenyltin (TPT), which have been used extensively as biocides in wood 
preservatives, in antifouling paints for boats and as pesticides. Mono- and di-substituted 
OTCs (dibutyltin, mono-n-octyltin and di-n-octyltin) are used as stabilizers in PVC plastics, 
and di-alkyltins have been approved as PVC stabilizers for food contact materials. OTCs 
tend to accumulate in fish and other aquatic organisms and tri-alkyltins are bio-degraded to 
di- and mono-alkyltin compounds and therefore these may be found also in addition to the 
tri-substituted OTCs. The structures of TBT and TPT are presented below. 





tributyltin (TBT) 

triphenyltin (TPT) 

Based on an EU SCOOP report the European Food Safety Authority estimated that the 
median concentrations of TBT, DBT and TPT in fish and fishery products are 7.0, 2.5 and 4.0 
ng/g product (EC, 2003; EFSA, 2004). The EU SCOOP report contains very few data on 
DOT, which were always below the limit of detection. The results for organotin in this 
study are given in table 3 at the end of this section. Organotin were found in three of the 
four samples that were analysed. The highest concentration of 9.0 ng/g was found for 
mono-butyl tin (MBT), a degradation product of TBT in the sample of tuna. Di-butyl tin 
(DBT) and TBT were also found in this sample. The pickled herring and the fish fingers 
contained butyl-tin as well as octyl-tin compounds. 

3.10 Result tables of the concentrations of determined parameters in 25 samples of 
food 

Tables 2 and 3 contain the full results of the study. Note that not all parameters are 
measured in all samples. In those cases the positions in the table are left blank. If 
concentrations of the parameters in a sample were below the detection limit, this is indicated 
with a "<" sign. The limits of detection for each parameter are printed directly after the 
compound name of the parameter. All results are expressed in ng/g product. 



Industrial Contaminants and Pesticides in Food Products 



81 



compound 
limit of detection 


-5 


1 


1 


s 

■f 

o 

s 


-..■ 

'-; 

Z 


-£ 


be 


£ 


c 
c 

Si 




1 


c 




n g/g 


n g/g 


ng/g 


ng/g 


ng/g 


ng/g 


ng/g 


n g/S 


n g/g 


"g/g 


n g/g 


n g/g 


n g/g 


ng/g 


Organochlorine pesticides 




























a-HCH 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


B-HCH 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


Y-HCH 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


HCB 0.1 


0.12 


0.22 


0.26 


0.18 


0.10 


0.34 


< 


< 


< 


0.10 


< 


0.10 


0.83 


a-chlordane 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


B-chlordane 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


o,p -DDE 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


p,p -DDE 0.1 


0.18 


0.48 


1.6 


0.43 


1.3 


0.79 


< 


< 


< 


0.25 


< 


< 


< 


o,p -DDD 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


p,p -DDD 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


o,p -DDT 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


p,p -DDT 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


Folychlorinated biphenyls 




























PCB18 0.1 


0.13 


0.15 


0.22 


0.16 


0.24 


0.25 


< 


0.12 


0.19 


0.16 


0.20 


0.26 


0.14 


PCB 28/31 0.1 


0.31 


0.22 


0.37 


0.36 


0.49 


0.56 


0.16 


0.36 


0.32 


0.35 


0.40 


0.44 


0.26 


PCB22 0.1 


< 


< 


0.11 


< 


0.14 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 41/64 0.1 


< 


< 


< 


< 


< 


0.27 


< 


< 


< 


< 


< 


< 


< 


PCB 44 0.1 


< 


< 


< 


0.18 


< 


0.27 


< 


< 


0.18 


0.24 


< 


0.18 


0.14 


PCB 49 0.1 


< 


< 


< 


< 


0.14 


0.18 


< 


< 


0.19 


0.13 


0.13 


0.16 


0.13 


PCB 52 0.1 


0.16 


< 


< 


0.15 


0.29 


0.40 


< 


0.21 


0.21 


0.16 


0.17 


0.27 


0.15 


PCB 54 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 56/60 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 70 0.1 


< 


< 


< 


0.12 


0.16 


0.20 


< 


< 


0.21 


0.12 


< 


0.17 


0.11 


PCB 74 0.1 


< 


< 


< 


< 


0.14 


0.22 


< 


< 


0.11 


< 


< 


< 


0.12 


PCB 87 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 90/101 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


0.11 


< 


< 


< 


PCB 99 0.1 


< 


< 


< 


< 


< 


0.14 


< 


< 


< 


< 


< 


< 


< 


PCB 104 0.1 


< 


< 


< 


< 


< 


< 


< 


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< 


< 


< 


< 


PCB 105 0.1 


< 


< 


< 


< 


< 


< 


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< 


< 


< 


< 


< 


< 


PCB 110 0.1 


< 


< 


< 


< 


< 


< 


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< 


< 


< 


< 


< 


< 


PCB 114 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 118 0.1 


< 


< 


< 


< 


< 


0.23 


< 


< 


< 


< 


< 


< 


< 


PCB 123 0.1 


< 


< 


< 


< 


< 


< 


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< 


< 


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< 


PCB 138 0.1 


< 


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< 


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< 


< 


< 


< 


PCB 141 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 149 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 151 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 153/168 0.1 


< 


0.22 


0.31 


0.16 


< 


0.29 


< 


< 


< 


0.12 


< 


0.14 


0.11 


PCB 156 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 157 0.1 


< 


< 


< 


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< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 158 0.1 


< 


< 


0.12 


0.10 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 167 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 170 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 177 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 180 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 183 0.2 


< 


< 


< 


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< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 187 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 188 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 189 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 194 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 199 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 203 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 



Table 2. Results for organochlorine pesticides and polychlorobipenyls expressed in ng/ g 
product. 



82 



Pesticides in the Modern World - Risks and Benefits 



compound 
limit of detection 


C 
1 


'— 

PL, 


| 


= 


t' 




■-'■ 
2 


bo 

— 


honey 
orange juice 
brown bread 




> 


n s/s 


ng/g 


n g/g 


n g/g 


n g/g 


n g/g 


n g/g 


n g/g 


n g/g 


ng/g ng 


/g ng/g 


n g/g 


Or gano chlorine pesticides 
























□-HCH 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< < < 


< 


S-HCH 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< < < 


< 


Y-HCH 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< < < 


< 


HCB 0.1 


0.14 


< 


0.10 


0.10 


< 


0.22 


< 


0.7 


< < < 


0.10 


a-chlordane 0.1 


< 


< 


< 


< 


< 


0.13 


< 


0.2 


< < < 


< 


P-chlordane 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< < < 


< 


o,p'-DDE 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 0.65 < 


< 


p,p'-DDE 0.1 


0.33 


0.17 


0.21 


0.17 


< 


0.83 


< 


5.6 


< 1.5 < 


0.40 


o,p'-DDD 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< < < 


< 


p,p'-DDD 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< < < 


< 


o,p'-DDT 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< < < 


< 


p,p'-DDT 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< < < 


< 


Poly chlorinated biphenyls 
























PCB 18 0.1 


0.14 


0.16 


0.22 


0.24 


0.45 


0.19 


0.20 


0.23 


0.38 


0.29 


< 


PCB 28/31 0.1 


0.36 


0.23 


0.59 


0.43 


0.63 


0.49 


0.38 


0.76 


0.55 


0.61 


0.19 


PCB 22 0.1 


< 


< 


0.14 


< 


0.13 


0.11 


< 


0.13 


0.11 


0.16 


< 


PCB 41/64 0.1 


< 


< 


0.14 


< 


< 


< 


< 


0.38 


< 


< 


< 


PCB 44 0.1 


0.24 


< 


0.29 


0.17 


0.22 


0.24 


0.19 


0.33 


0.19 


0.21 


< 


PCB 49 0.1 


< 


0.12 


0.19 


0.11 


0.21 


0.23 


< 


0.22 


0.15 


0.19 


< 


PCB 52 0.1 


0.25 


0.20 


0.28 


0.24 


0.27 


0.36 


< 


0.74 


0.19 


0.35 


< 


PCB 54 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 56/60 0.1 


< 


< 


< 


< 


< 


0.18 


< 


0.31 


< 


0.14 


< 


PCB 70 0.1 


0.13 


< 


< 


< 


0.14 


0.38 


< 


0.71 


0.16 


0.17 


< 


PCB 74 0.1 


< 


< 


< 


< 


< 


0.19 


< 


0.37 


< 


0.11 


< 


PCB 87 0.1 


< 


< 


< 


< 


< 


0.12 


< 


0.41 


< 


< 


< 


PCB 90/101 0.1 


< 


< 


0.13 


< 


< 


0.47 


0.14 


2.4 


< 


0.15 


< 


PCB 99 0.1 


< 


< 


< 


< 


< 


0.24 


< 


0.95 


< 


< 


< 


PCB 104 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 105 0.1 


< 


< 


< 


< 


< 


0.14 


< 


0.59 


< 


< 


< 


PCB 110 0.1 


< 


< 


< 


< 


< 


0.30 


0.14 


1.6 


< 


< 


< 


PCB 114 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 118 0.1 


0.11 


< 


< 


< 


< 


0.40 


0.11 


1.8 


< 


< 


< 


PCB 123 0.1 


< 


< 


< 


< 


< 


< 


< 


0.27 


< 


< 


< 


PCB 138 0.1 


< 


< 


0.10 


< 


< 


0.24 


0.20 


1.5 


< 


< 


< 


PCB 141 0.1 


< 


< 


< 


< 


< 


< 


< 


0.30 


< 


< 


< 


PCB 149 0.1 


< 


< 


< 


< 


< 


0.49 


< 


2.8 


< 


< 


< 


PCB 151 0.1 


< 


< 


< 


< 


< 


0.17 


< 


0.92 


< 


< 


< 


PCB 153/168 0.1 


0.26 


< 


0.13 


< 


< 


1.0 


0.12 


5.5 


< 


0.13 


< 


PCB 156 0.1 


< 


< 


< 


< 


< 


< 


< 


0.22 


< 


< 


< 


PCB 157 0.1 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 158 0.1 


< 


< 


< 


< 


< 


0.50 


0.16 


3.2 


< 


< 


< 


PCB 167 0.1 


< 


< 


< 


< 


< 


< 


< 


0.19 


< 


< 


< 


PCB 170 0.2 


< 


< 


< 


< 


< 


< 


< 


0.56 


< 


< 


< 


PCB 177 0.2 


< 


< 


< 


< 


< 


< 


< 


0.56 


< 


< 


< 


PCB 180 0.2 


< 


< 


< 


< 


< 


0.32 


< 


0.90 


< 


< 


< 


PCB 183 0.2 


< 


< 


< 


< 


< 


< 


< 


0.43 


< 


< 


< 


PCB 187 0.2 


< 


< 


< 


< 


< 


< 


< 


1.6 


< 


< 


< 


PCB 188 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 189 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 194 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 199 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


PCB 203 0.2 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 


< 



Table 2. (continued) Results for organochlorine pesticides and polychlorobipenyls expressed 
in ng/g product. Empty spaces, as PCBs in orange juice, indicate that the parameter was not 
determined in this sample. 



Industrial Contaminants and Pesticides in Food Products 



83 





n s/g 


Brominated flame retar dents 


BDE-17 


0.1 


BDE-28 


0.1 


BDE-32 


0.05 


BDE-35 


0.05 


BDE-37 


0.05 


BDE-47 


0.05 


BDE-49-71 


0.05 


BDE-66 


0.05 


BDE-75 


0.05 


BDE-77 


0.05 


BDE-85 


0.05 


BDE-99 


0.05 


BDE-100 


0.05 


BDE-119 


0.05 


BDE-126 


0.05 


BDE-138 


0.05 


BDE-153 


0.05 


BDE-154 


0.05 


BDE-156 


0.05 


BDE-166 


0.05 


BDE-181 


0.05 


BDE-183 


0.05 


BDE-184 


0.05 


BDE-190 


0.1 


BDE-191 


0.1 


BDE-196 


0.2 


BDE-197 


0.2 


BDE-206 


1 


BDE-207 


1 


BDE-209 


5 


Per flu urinated chemicals 


PFOA 


0.2 


PFNA 


0.2 


PFDA 


0.2 


PFUnA 


0.2 


PFDoA 


0.2 


PFTrA 


0.2 


PFOS 


0.2 


PFOSA 


0.2 


Phthalates 




DMP 


1 


DEP 


1 


DIBP 


10 


DIM' 


10 


13 131' 


1 


DEHP 


20 


DINP 


2(1 


DIDP 


20 


Alkylphenols 




NP 


2 


OP 


2 


Artificial musks 


AHTN 


0.1 


HHCB 


0.1 


MK 


0.1 


MX 


0.1 


Organotin con 


lpounds 


MBT 


0.2 


DBT 


0.2 


TBT 


0.2 


MOT 


0.2 


DOT 


0.2 



ng/g ng/g ng/g ng/g ng/g ng/g ng/g 



0.07 < 0.06 



0.75 0.3 0.43 0.82 0.29 



0.15 

0.12 



ng/g ng/g ng/g ng/g 



0.06 < 0.08 0.06 0.06 



0.27 0.41 0.26 0.33 0.26 



< < < < < 5.6 

< < 4400 < 1500 < 

< 76 190 200 780 132 
21 32 50 9.9 25 17 
910 3000 210 130 890 770 
31 26 59 < 660 < 



1320 640 670 20 210 



Table 3. Results for brominated flame retardants, perfluorinated chemicals, phthalates, 
alkylphenols, artificial musks and organotin compounds expressed in ng/g product. Empty 
spaces, as for the artificial musks, indicate that the parameter was not determined in this 
sample. 



84 



Pesticides in the Modern World - Risks and Benefits 





ng/g 


n s/s 


n g/g 


Bro initiated flame retardents 






BDE-17 


0.1 


< 


< 


BDE-28 


0.1 


< 


< 


BDE-32 


0.05 


0.07 


0.05 


BDE-35 


0.05 


< 


< 


BDE-37 


0.05 


< 


< 


BDE-47 


0.05 


0.78 


0.52 


BDE^9-71 


0.05 


< 


< 


BDE-66 


0.05 


< 


< 


BDE-75 


0.05 


< 


< 


BDE-77 


0.05 


< 


< 


BDE-85 


0.05 


< 


0.32 


BDE-99 


0.05 


0.12 


< 


BDE-100 


0.05 


0.11 


< 


BDE-119 


0.05 


< 


< 


BDE-126 


0.05 


0.08 


< 


BDE-138 


0.05 


< 


< 


BDE-153 


0.05 


< 


< 


BDE-154 


0.05 


< 


< 


BDE-156 


0.05 


< 


< 


BDE-166 


0.05 


< 


< 


BDE-181 


0.05 


0.18 


< 


BDE-183 


0.05 


< 


< 


BDE-184 


0.05 


< 


< 


BDE-190 


0.1 


< 


< 


BDE-191 


0.1 


< 


< 


BDE-196 


0.2 


< 


< 


BDE-197 


0.2 


< 


< 


BDE-206 


1 


< 


< 


BDE-207 


1 


< 


< 


BDE-209 


5 


< 


< 


Perf luorinated chemicals 






PFOA 
PFNA 
PFDA 


0.2 
0.2 
0.2 






PFUnA 


0.2 






PFDoA 


0.2 






PFTrA 


0.2 






PFOS 


0.2 






PFOSA 


0.2 






Phthalates 








DMP 


1 


< 


< 


DEP 


1 


< 


< 


DIBP 


10 


< 


< 


DBP 


10 


< 


170 


BBP 


1 


< 


13 


DEHP 


20 


290 


140 


DINP 


20 


< 


470 


DIDP 


20 


< 


< 


Alkylphenols 








NP 


2 


< 


< 


OP 


2 


< 


< 


Artificial musks 






AHTN 


0.1 






HHCB 


0.1 






MK 


0.1 






MX 


0.1 






Organotin con 


lpounds 






MBT 


0.2 






DBT 


0.2 






TBT 


0.2 






MOT 


0.2 






DOT 


0.2 







ng/g ng/g ng/g 



ng/g ng/g ng/g ng/g ng/g ng/g 



0.08 0.15 



0.39 0.35 



0.18 0.29 
0.27 0.56 



Table 3. (continued) Results for brominated flame retardants, perfluorinated chemicals, 
phthalates, alkylphenols, artificial musks and organotin compounds expressed in ng/g 
product. Empty spaces, as for the brominated flame retardants in orange juice, indicate that 
the parameter was not determined in this sample. 



Industrial Contaminants and Pesticides in Food Products 85 

4. Conclusions 

In this study the concentrations of a number of typical man-made chemicals in food or food 
products were determined. The compound groups of interest were organochlorine 
pesticides, polychlorinated biphenyls, brominated flame retardants, phthalates, 
alkylphenols, artificial musks, perfluorinated compounds and organitin compounds. The 
results show that many of these compounds are present food in the range of 0.1 to 10 ng/g 
with the exception of phthalates for which the typical concentrations are two orders of 
magnitude higher. Organochlorine pesticides were found in the 17 of the 25 samples. The 
main organochlorine pesticides found in food are p,p'-DDE, a metabolite of DDT, and HCB 
in concentrations up to 5.6 ng/g. Polychlorinated biphenyls were found in all samples with 
predominance for PCB-18, -28 and -52. The sum of the indicator-PCBs ranged from 0.16 to 
13 ng/g and total PCBs up to 32 ng/g. The highest concentrations were found in fish. 
Brominated flame retardants were found in 19 of the 24 samples with predominance for the 
tetra- and penta-PBDEs, especially BDE-47, -32 and -99. Total PBDE concentrations ranged 
from 0.15 to 1.2 ng/g with the highest concentration found in meat and not in fish as in 
other studies. BDE-209, HBCD and TBBPA were not found in any of the samples. The 
prefluorinated compounds PFOS and PFOSA were found in one of the four samples 
analysed, a fish sample, in concentrations of 1.3 and 0.2 ng/g. The predominant phthalates 
in food were DEHP, DBP and BBP. Phthalates were found in 12 of the 19 samples. DEHP 
concentrations ranged from 20 to 24,000 ng/g, the latter for a sample of olive oil, with a 
median concentration of 640 ng/g. Median concentrations for DBP and BBP were 200 and 17 
ng/g. Alkylphenols were detected in 2 of the 19 samples, in both cases nonylphenol in 
concentrations around 5 ng/g. Of the artificial musks the poly cyclic musks HHCB and 
AHTN were found 2 of the 4 samples in concentrations up to 0.56 ng/g for HHCB. As in 
other matrices the AHTN concentrations are about half those of HHCB. Organotin 
compounds were found in three of the five samples. Apart from TBT and its metabolites 
DBT and MBT, two samples also contained octyltin compounds. 

5. Acknowledgement 

We kindly acknowledge the support of the WWF in the execution of this study. 

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Pesticide Residues in Bee Products 

Emmanouel Karazafiris 1 , Chrysoula Tananaki 1 , 
Andreas Thrasyvoulou 1 and Urania Menkissoglu-Spiroudi 2 

laboratory of Apiculture-Sericulture, Faculty of Agriculture, 

Aristotle University of Thessaloniki, 

2 Pesticide Science Laboratory, Faculty of Agriculture, 

Aristotle University of Thessaloniki, 

Greece 



1. Introduction 

"The man has no divine right over the food. He must compete for this with weeds, diseases, 
insects and other organisms" (Grodner, 1996). 

With the above quote, Grodner clearly reflects the situation in the area production and 
processing of food. More than 10.000 species of insects and mites, 1.500 species of fungi and 
600 plant species have been identified as harmful to agriculture (Grodner, 1996; Pimentel et 
al., 2000). The production of plant and animal products requires the use of large quantities 
of chemicals (plant protection agents, veterinary drugs, fertilizers, etc.), which could lead to 
increased production and improved quality, as a consequence. The quality of the final 
product is usually reflected to particular visual parameters like color, size and general 
appearance. In the case of food, however, many questions are raising about their safety. The 
reason is the possible presence of chemical residues detected in the final product. 
Nowadays, consumer safety is a major priority for governments of developed countries and 
food safety is a criterion for the trading and prices on the market. Reports in media about 
alimentary scandals cause anxiety to consumers and turning a part of the market to organic 
products, which are considered free, or at least less contaminated by hazardous substances. 
In recent years, reports in media have grown at an alarming rate and any references to the 
consumer aimed at creating impressions, achieved by overemphasizing the disadvantages 
of chemical use and mainly problems related to environmental pollution and its impact on 
human health. In contrast, reports in media referring to the advantages of using chemicals 
are minimal to nonexistent. For example, the absence of appropriate chemical agents to 
combat rodents lead to the first epidemic of the bubonic plague disease and the death of 
65.000.000 people. Moreover, the starvation in Ireland began due to the fungal disease 
Phytophthora infestans, which destroyed potatoes causing 1.000.000 deaths from 1845 to 1851 
(Knutson et al., 1990). Besides health problems that preoccupied humans in the past, the 
economic impact on different groups of consumers will be significant in case of pesticides 
withdrawal. Specifically, the weekly expenses for food are expected to rise by 44% for 
consumers of low average income. The economies of countries with intensive agriculture 
will be stroke because of the decline in exports of grains and products like cotton. Finally, 
undesirable environmental effects are expected due to the increasing of cultivated land and 
the erosion problems that could be observed because of the limited growth of the root 



90 Pesticides in the Modern World - Risks and Benefits 

system of plants (Knutson et al., 1990). In contrast to the above, there are few cases of 
pesticides proved dangerous to public health. A typical example is the chlorinated 
hydrocarbon DDT, which was previously used against mosquitoes. The active substance 
DDT contributed greatly to reduce the spread of diseases like malaria, but was withdrawn 
in 1970 as it was considered dangerous for human and environment safety. 
Active substances are classified into five groups according to their toxicity. The first group 
(la) includes the extremely toxic agricultural plant protection agents, while the four other 
groups of substances are listed in order of decreasing toxicity (lb, II, III). The fifth group (U) 
includes substances that are unlikely to become toxic to humans (International Programme 
on Chemical Safety, 2004). The adverse effects of these compounds may be observed in a 
short period (acute toxicity) or after a long time (chronic toxicity). In any case, it should be 
noted that, although the annual reported number of deaths by poisoning is 355.000, only a 
part of these poisonings, which is not specified in the World Health Report, are due to 
pesticides (WHO The World Health Report, 2003). Moreover, all cases of poisoning are due 
to accidents, like accidental ingestion or inhalation of chemicals, and not by food intake. 
However, the possibility of indication of various effects in consumer health through chronic 
toxicity cannot be ignored. Toxicity of a plant protection product depends on various 
factors, including chemical structure, temperature and humidity conditions, dose, duration 
of exposure, mode of action and the kind of exposure, like ingestion, inhalation, dermal etc. 
Different groups of pesticides and veterinary drugs are likely to be responsible for causing 
malaise, sore eyes, abnormalities to skin and respiratory system. Moreover, some pesticides 
and veterinary drugs are suspected of causing certain types of cancer, teratogenicity, 
chromosomal abnormalities and the weakening of the immune system of humans (Banerjee, 
1999). The toxicity of various active substances, which can be detected in bee products, 
varies according to their chemical synthesis. In any case, poisoning or deaths due to the 
presence of toxic substances exclusively to bee products have not been reported. An 
exception is the death of infants, which was caused by Clostridium botulinum (Arnon, 1980). 
Even in these cases, however, the responsibility of honey has not been proven clearly, as this 
Clostridium appears in the environment widely (Midura, 1996). In addition, 65% of infants 
that became ill had not eaten honey at all (Arnon et al., 1979). In any case, appropriate infant 
feeding prohibits the consumption of honey until the age of one year to eliminate possible 
poisoning from toxins of this micro-organism. 

Another way of classification of active substances, relates to the subject of this investigation, 
and is based on bee toxicity. In general, active substances are classified as very toxic, 
moderately toxic and non-toxic to bees. The most significant impact on bee colonies has 
been observed after treatments with plant protection products during the blooming. Most 
deaths occurred during the stage of forager worker bee that collects nectar and pollen. 
Moreover, larvae and domestic bees die because of pesticide residues detected in pollen. 
Although in many cases the concentration of pesticide found in pollen is not lethal, it is 
however likely to cause paralysis of bees, irritability, killing and replacing of the queen bee 
and generally abnormal behavior. This behavior can also be caused by substances that do 
not kill bees directly (e.g. carbaryl as active ingredient of Sevin®) but are transferred into the 
hive by foragers and affect the entire population (Sanford, 1993). The long-term persistence 
of many pesticides in stored pollen has also serious impact of bees' survival. Arsenic from 
paris green and calcium arsenate was present in pollen stored in comb analyzed six months 
after application. Methomyl residues persisted in honeybee combs for eight months. Methyl 
parathion from Penncap M, persisted in combs samples of stored pollen for 7 to 14 months 
after use and carbaryl similarly persisted over winter for 7-9 months (Erickson et al., 1983) 



Pesticide Residues in Bee Products 91 

Neonicotinoids like imidacloprid were also detected in stored pollen (Gregorc & Bozic, 2004; 
Chauzat et al., 2006). Also, the type of formulation and application of the pesticide in 
relation to toxicity caused to the bees proved particularly important. For example, the 
standardization of the active ingredient methyl parathion in microcapsules kills 13 times 
more bees than the formulation of the same substance as an emulsified solution. 
Furthermore, wettable powder or dust proved less dangerous than microcapsules, but also 
caused more deaths than aqueous or emulsified solutions (Sanford, 1993). 
Under the development of framework concerning consumer safety, the European Union 
created a warning system called RASFF (Rapid Alert System for Food and Feed), which 
reports hazardous foods and feeds, identified in the markets of the Member States. Bee 
products like honey and royal jelly have been reported occasionally. The main reason why 
they have been reported is the detection of residues of antibiotics that have been used by 
beekeepers to fight various diseases of bees. 

2. Contamination of bee products 

There are two ways of contamination of bee products with various chemicals; the indirect 
and the direct contamination. The indirect way reflects the transporting of toxic substances 
by foragers bees during the collection of nectar, honeydew, water, pollen and propolis. 
Many studies concern the contamination of hive products by agrochemicals and heavy 
metals, while few concern the presence of nitrofurans, toxins and PCBs in beehive products. 
The direct way, which is the most important, regards the contamination of bee products by 
acaricides, antibiotics and volatile pesticides caused by beekeeping practices. 

2.1 Indirect contamination of bee products 

Many researchers supported the theory that the transferring of pesticides from fields to 
beehive is prevented in various ways. The bees' death at the field, the lost of orientation of 
the foragers, the reluctance of guard bees to permit the entrance to foragers with 
contaminated nectar, the retaining of contaminated food in bees' stomach, the stopping of 
further elaboration of contaminated nectar by hive bees and the removal of affected bees 
from the hive are natural provisions against general contamination of honey (Johansen & 
Mayer, 1990; Atkins, 1992). Contrary to the above-mentioned cases, older studies reported 
that worker bees may carry high concentrations of pesticides into their beehive. In some 
cases the concentration of pesticides in the load was 25 times greater than the lethal dose of 
the bee (Jaycox, 1964). 

2.1.1 Pesticides 

Pesticides used on various crops are classified into groups based on their chemical structure 
(organophosphates, pyrethroids, organochlorines, carbamates, neonicotinoids etc.), mode of 
action (systemic, contact), target (insecticides, acaricides, herbicides, fungicides, bactericides, 
nematicides) and synthesis (synthetic or natural). The residues of pesticides detected in 
beehive products are classified in the groups of insecticides (organochlorines, 
organophosphates, carbamates and neonicotinoids), acaricides, fungicides and herbicides. 

2.1.1.1 Organochlorine pesticides (OCPs) 

This specific group of insecticides is considered particularly hazardous because of its ability 
to bioaccumulate into the food chain, to remain stable for many years and to move into the 



92 Pesticides in the Modern World - Risks and Benefits 

environment in every potential way (air, water, soil, biota). The case of bioaccumulation of 
DDT in the environment is the most characteristic, while the concentration detected in the 
higher levels of the food chain is 10,000,000 times greater than that detected into the water. 
In recent decades there had been many efforts worldwide to prevent the use of substances 
belonging to the group of persistent organic pollutants (POP), which includes many 
organochlorine compounds. The continuous transfer of semi-volatile compounds from 
tropical regions of the world to the colder poles is suspected for long-term effects on living 
beings (Carson R., 1962). Chlorinated hydrocarbons are detected in high concentrations in 
various products, because of their low rate of degradation. Wax is the beehive product more 
likely to be contaminated by organochlorine insecticides because of its strong lipophilic 
character. Moreover, OCPs were proved to remain stable during the conversion of old 
combs into new (Jimenez et al., 2005). The problem is magnified by the import of wax from 
continents where the use of chlorinated hydrocarbons is still permitted like Asia and Africa. 
The encouraging news is that the percentage of honey contaminated with chlorinated 
hydrocarbons dropped from 96.1% to 52.3% during the decades 1980 and 1990. 

2.1.1.2 Organophosphorus pesticides (OPPs) 

This specific class of pesticides is of relatively high toxicity for humans and was first studied 
and used as an asphyxiating gas during the Second World War. Organophosphorus 
compounds are not stable in the environment and are not bio-concentrated and this is 
probably the main reason why they were detected rarely and at lower concentrations into 
beehive products. Most of published information concerns the compound methyl parathion, 
which has been used in agricultural crops as preparation in the slow-release form of 
microcapsules. Residues of this chemical were detected in honey and pollen (Atkins & 
Kellum, 1984). The microcapsules stick on the dense coat of bees, transferred into their hive 
and stored along with pollen. Pollen is the main component of the diet of larvae. The 
presence of polluted pollen might cause poisoning and eventually death to brood of bees. In 
a survey conducted on pollen from France, residues of parathion and methyl parathion were 
found in 1.2% and 4.9% of the samples, respectively. The average concentration for 
parathion was 0.019 mg kg- 1 and for parathion methyl was 0.025 mg kg- 1 (Chauzat et al., 
2006). Blasco et al. (2003) detected only heptenophos in 4% of honey samples that were 
analyzed, out of 23 organophosphorus pesticides that they researched. Heptenophos 
concentrations in this survey ranged from 0.08 mg kg- 1 to 0.23 mg kg -1 . Finally, Balayiannis 
and Balayiannis (2008) detected the organophosphorus compounds chlorfenvinphos, 
chlorpyriphos and phorate in honey originated from Greece, in concentrations ranged from 
0.7 pg kg- 1 to 0.89 ug kg- 1 . 

2.1.1.3 Carbamate pesticides 

Carbamate insecticides have a similar mode of action with organophosphates but their 
insecticidal activity is more selective and depends to a certain extent on the insect species. 
Some fungicides and herbicides belong to this family. These substances are highly volatile in 
the environment and in some cases they were detected in beehive products. Concentration 
of carbamate residues detected in pollen ranged from 0.126 mg kg- 1 to 0.265 mg kg- 1 for the 
active ingredient carbaryl, while the maximum concentration of carbofuran was 0.14 mg kg- 1 
(Chauzat et al., 2006). Concentration of carbaryl, carbofuran, pirimicarb and methiocarb 
residues, in most cases is considered low and does not exceed 0.071 mg kg- 1 . In only one 
Spanish honey the concentration of carbofuran was 0.645 mg kg- 1 (Blasco et al., 2003). 



Pesticide Residues in Bee Products 93 

Nevertheless, the concentration of carbamate residues is low in pollen and honey, while no 
residues have been reported in other beehive products. 

2.1.1.4 Neonicotinoid pesticides 

Most studies reported on neonikotinoids insecticides, refer to the active ingredient 
imidacloprid. This substance was proved toxic to bees, but the concentrations of residues 
detected in honey were very low (0.001 mg kg- 1 to 0.005 mg kg- 1 ) (Bonmatin et al., 2003; 
Maus et al., 2003; Schmuck et al., 2001). In many cases residues did not exceed the limit of 
quantification (<0.002 mg kg- 1 ) (Rogers & Kemp, 2003; Schoning & Schmuck, 2003; Stadler et 
al., 2003; Faucon et al., 2004). Detected residues of imidacloprid in pollen were 0.005 mg kg- 1 , 
while detection rate was 49.4% (Bonmatin et al., 2003). The hazard quotient (application rate 
in grams per hectare/ LD 50 ) of neonicotinoids is far below the trigger value of 50, but the 
most important is the chronic toxicity that they cause to bees. The long-term exposure to 
neonicotinoid after the behaviour of bees, reduce their reproduction capacity and lead of 
population decline. The low detectable concentrations in combination with the low toxicity 
of imidacloprid in humans are reassuring for consumer safety. On the contrary, the effects of 
imidacloprid residues on bees should be further explored. The high toxicity for bees makes 
neonicotinoid residues suspicious about the death of many forager bees collecting nectar 
from sunflower, corn and cotton crops. The implementation of neonicotinoid active 
substances in seed of plants like cotton, corn and sunflower led to a theory that this specific 
class of pesticides is responsible for the "colony collapse disorder" syndrome (CCD). The 
CCD is defined as the sudden depopulation of a beehive and the rapid collapse of the 
colony. The causes of this phenomenon are not clear yet. The suspicion is directed at the 
mite Varroa destructor Anderson & Trueman, while others blame the protozoan Nosema 
ceranae. Additionally, suspicion directed at poisoning of bees by neonicotinoid insecticides 
and at various forms of radiation (telephony, wireless networks etc.) as well. In fact, 
research on the toxicity of neonicotinoids to the bee, proved tolerance of the insect body at 
normal concentrations identified in honey, pollen and nectar (Schmuck et al., 2001; Faucon 
et al., 2004). More recent research is directed at the effect of imidaclorpid to the orientation 
of bees (Bortolotti et al; 2003). 

2.1.1.5 Fungicides 

Fungicides are toxic substances that are used to kill or inhibit the growth of fungi that 
cause economic damage to crops and endanger the health of domestic animals or humans. 
Most fungicides are toxic to humans and can cause both acute and chronic problems if 
absorbed into food. Kubik et al. (1999; 2000) studied the possibility of contamination of 
beehive products with residues of chemicals used on apple and cherry trees. Vinclozolin, 
iprodione and thiophanate methyl residues were detected in honey and pollen collected 
from cherry flowers, while captan and difenuconazole were detected in beehive products 
collected from apple trees. Specifically, the average concentration levels of vinclozolin in 
honey were determined at 0.107 mg kg- 1 A recent review of vinclozolin by the US 
Environmental Protection Agency has concluded that the chemical or its breakdown 
products are associated with the development of testicular tumors in rats. The mean 
concentrations of residues of other active compounds were 0.0006 mg kg- 1 , 0.009 mg kg- 1 , 
0.023 mg kg- 1 and 0.059 mg kg- 1 for captan, difenuconazole, iprodione and methyl 
thiophanate respectively. In all cases, the concentration was lower in honey, than in stored 
pollen (Kubic, 2000). 



94 Pesticides in the Modern World - Risks and Benefits 

2.1.2 Antibiotic residues due to agricultural use 

Antibiotics can find their ways to bee products not only from beekeepers but also from the 
environment. Bees collect and transfer readily in their hive bactericides that are used against 
Envinia amylovora. Out of 166 Greek citrus honeys that had been analyzed the 146 of them 
were found having antibiotic residues of soulphonamides and streptomycine originating 
from the therapeutical products that had been used in citrus plants (Karampournioti, 2004). 
Similarly in South Germany 40 samples out of 183 (21%) were found having residues of that 
source (Wallner, 1998). Moreover, Brasse (2001) identified the antibiotic streptomycin in 27 
out of 128 honey analyzed samples. Bees may also transfer antibiotics through water since 
sulphanimide and tetracyclines are used in drinking water from poultry farms, rabbit cages 
and other animals. The manure of pigs and cows treated with sulphonamides or sulpha- 
compounds could also be the vector. Some herbicides products, like Asulan may be 
degradated to sulphanilamide and bees with nectar can transfer it into the hive (Bogdanov 
& Edder, 2004; Kaufmann & Kaenzig, 2004). Finally bees may rob honey from colonies of 
other apiary that had been treated by antibiotics and by this way can contaminate their 
product in detectable levels. 

2.2 Direct contamination of bee products 

Active substances used by beekeepers themselves are likely to contaminate bee products 
with undesirable residues. Acaricide and antibiotic preparations are used in order to control 
the mite Varroa destructor, American foulbrood, Nosemosis and other diseases. Moreover, 
several volatile insecticides were used in the warehouse, in order to fend lepidopteron 
Galleria mellonella Linnaeus, which is responsible for considerable damages to stored combs. 

2.2.1 Acaricide residues 

The use of synthesized substances for crop protection and livestock is the easiest and most 
effective way for beekeepers to control mites. Acaricides like amitraz, cymiazole, 
bromopropylate, tau-fluvalinate, flumethrin, coumaphos and malathion have been used by 
beekeepers all over the world. Many preparations like Apistan (a.i. tau-fluvalinate), Perizin 
(a.i. coumaphos), CheckMite+ (a.i.coumaphos), Bayvarol (a.i. flumethrin) and Apiguard (a.i. 
thymol) gain approval in most European countries. There are substances like amitraz that 
got approval only in certain countries and others like malathion that have not been 
approved at all. 

2.2.1.1 Amitraz 

Structure: It belongs to the group of formamidines 

Action: Non-systemic insecticide and acaricide, which causes stimulation of neuronal 

activity killing the target. 

Preparation: The main commercial formulation is the Taktik, used in livestock and 

particularly horses and sheep. Other preparation used: Mitak and Bye Bye. 

Ways to use in beekeeping: Fumigation, Spray. 

Acceptable Daily Intake (ADI): 0.003 mg kg- 1 body weight per day or 0.18 mg per person per 

day (EMEA, 1999). 

MRL for honey: EU established maximum residue levels (MRL) for amitraz residues in 

honey. The MRL of amitraz established to 0.2 mg kg -1 including the parent compound and 

its metabolites containing 2,4-dimethylaniline moiety. It should be noted that despite the 

establishment of the MRL, amitraz residues in honey are not acceptable in some countries 



Pesticide Residues in Bee Products 95 

because of the lack of approval for beekeeping use. Therefore, in this case the limit 
corresponds to the Limit of Quantification, which is 0.01 mg kg- 1 . 

The use of active substance amitraz is widespread in several European countries and the 
United States. Moreover, the effectiveness of this substance against varroa is satisfactory. 
The residues of the active substance is not often detected because of the rapid degradation of 
amitraz, which takes place within three weeks in blossom honey and four weeks in 
honeydew honey. The difference in degradation interval was attributed to the lower pH of 
blossom honey, which accelerates the chemical reactions of decomposition (Corta et al., 
1999). The active ingredient amitraz is usually detected in cases where the preharvest 
interval is very short. A study reports as final degradation product of amitraz in honey, the 
2,4-dimethyl-aniline, which is classified as hazardous to public health (Taccheo et al., 1988a). 
Recently, several samples of pears found to contain significant concentrations of amitraz 
and its metabolites. This fact, as well as indications about carcinogenic effects of the 
substance, led to a series of inspections and repeated alerts reported on RASFF of EU (Rapid 
Alert System of Food And Feed). To date, no published RASFF on residues of amitraz in 
honey have been reported. Finally, a reference work published in USA, observed the 
development of resistance of varroa to amitraz (Eljen et al., 2000). 

2.2.1.2 Coumaphos 

Structure: It belongs to the group of organophosphorus insecticides-acaricides 

Action: Substance with systemic action that causes death in insects and mites by affecting 

cholinergic synapses of the central nervous system. 

Preparations: The active ingredient coumaphos prepared by Bayer as three different 

formulations; Perizin, CheckMite+ and Asuntol. The last is the only one without 

authorization for beekeeping use. 

Ways to use in beekeeping: The active substance used as an aqueous solution or a controlled 

release film. Perizin is used as an aqueous solution applied as drops between the frames. 

Spraying or adding to food can also be used for the application of this preparation. Special 

mention should be made to the use of coumaphos in the form of controlled release strips 

(CheckMite+). Primarily, application of CheckMite+ took place in the U.S., by providing a 

limited number of films in beekeepers of every State (Sanford & Flottum, 1999). In Europe, 

CheckMite+ was granted authorization in 2006. The major advantage of this preparation is 

that it also controls the small hive beetle Aethina tumida Marey. 

ADI: 0.25 mg kg- 1 body weight per day or 15 mg per person per day (EMEA, 2001). 

MRL for honey: the established MRL for coumaphos in the EU is 0.1 mg kg- 1 . 

Coumaphos does not control mites exclusively through contact, like most acaricides used in 

beekeeping, but it has a systemic action as well. The advantage of this way of action is the 

greater efficacy, and the rapid dispersion throughout the whole area of the hive. However, 

the disadvantage of substances with systemic action like coumaphos is the great persistence. 

According to a study, bees produce wax with residues of coumaphos, even six months after 

the application of the substance into the hive (Wilhelmina, 1992). 

The persistence and dispersion of coumaphos in the hive after the application of 

CheckMite+ was studied by Karazafiris et al. (2008). According to that study, concentration 

of coumaphos residues was great in honey frames, which were in contact with strips. In 

some cases, residues exceeded the value of the established MRL. Moreover, it was observed 

that the concentration of acaricide in honey was at the level of MRL even 103 days after the 

removal of the strips. Therefore, the exclusion of frames that are in contact with the strips 



96 Pesticides in the Modern World - Risks and Benefits 

could lead to a drastic reduction of residual coumaphos concentrations in the final product. 
On the contrary, it was observed that the concentration of coumaphos residues in honey 
chamber was significantly lower and in no case exceeded the MRL. Finally, the time 
between application of the preparation and the collection of honey affected the amount of 
residues. Gajduskova et al. (1990), studied the contamination of bee products under 
different methods of application, found that higher concentrations of coumaphos levels 
were recorded when the substance was added to the syrup rather than the usual method of 
dripping the chemical into the hive. Coumaphos proved very stable in honey, moved 
quickly to the wax because of its strong lipophilic character and remained there in 
significant concentrations even after melting of the wax (Krieger, 1991). Reports that mites 
became resistant to coumaphos have already been published (Maggi et al., 2009; Petis, 2004). 

2.2.1.3 Flumethrin 

Structure: It belongs to the group of pyrethroids. 

Action: Non-systemic insecticide-acaricide which acts in contact through the stomach. As 

the majority of pyrethroid pesticides, flumethrin is characterized as a broad range pesticide 

presenting low toxicity to mammals. Through its action, flumethrin disrupts the functioning 

of the Na+ pump and therefore the equilibrium of Na + /K + across the membrane. 

Preparation: Bayvarol is one of the approved preparations for use in beekeeping. Production 

Company is the Bayer CropScience. 

Beekeeping use: Flumethrin applied in the form of controlled release strips. 

ADI: 1.8 mg kg- 1 body weight per day or 108 mg per person per day (EMEA, 1998). 

MRL for honey: The very low concentration required per hive and the low water solubility, 

are the main reasons why no detectable residues were detected in honey after the 

recommended use. That is the reason why no MRL has been established for this substance 

(EMEA, 1998). According to recent studies, mites became resistant to pyrethroids (Milani, 

1995; Thompson, 2003). 

2.2.1.4 Tau f luvalinate 

Structure: It belongs to the group of pyrethroids. 

Action: This is a broad range non-systemic insecticide-acaricide that acts by contact through 

stomach. The way of action of tau fluvalinate is similar to that of the flumethrin. 

Preparation: There are three preparations used by beekeepers containing tau fluvalinate: 

Apistan, Mavrik and Klartan. Out of the three, only Apistan has an approval for beekeeping 

use. 

Beekeeping use: The use of the authorized preparation is in the form of controlled release 

strips (Apistan). 

ADI: 0.5 mg kg- 1 body weight per day or 30 mg per person per day (EMEA, 1998). 

MRL for honey: EU established no MRL for tau fluvalinate residues in honey, as the 

concentrations of detected residues were extremely low, based on the experimental results 

included in the file submitted (<0.01 mg kg- 1 ) (EMEA, 1998). 

The use of Apistan strips is likely to lead to accumulation of residues, if they have been left 

in the hive for more than 6 weeks. Balayiannis and Santas (1989) reported an increased 

persistence of residues in stored honey, compared with the honey in combs. This is 

apparently owed to the non-transfer of tau fluvalinate in wax. Tau fluvalinate is the most 

lipophilic of all compounds used in beekeeping. This property combined with the high 

stability of the substance in the wax contributes to the drastic increase in the concentration 



Pesticide Residues in Bee Products 97 

of residues in the honeycombs (Tsigouri et al., 2004). Tau fluvalinate has been used by 
beekeepers for many decades. Nowadays, the beekeepers have stopped using it because the 
mites developed resistance towards this chemical. (Elzen et al., 2000; Milani et al., 1995; 
Thompson et al., 2003). 

2.2.1.5 Bromopropylate 

Bromopropylate is one of the oldest compounds that had been used against Varroa under 

the commercial product FOLBEX-VA. Its use was totally abandoned in Switzerland in 1991. 

An analysis made 19 years later showed that bromopropylate was still in beeswax in high 

concentrations and scientists believe that more than 20 years will pass before it is expected 

to fully disappear from beeswax. 

Structure: It belongs to the group of chlorinated derivatives of benzene. 

Action: bromopropylate is a broad spectrum, non-systemic insecticide-acaricide with high 

residual activity, which acts by contact and inhibits the synthesis of ATP. 

Preparations: The name of the commercial preparation is Folbex VA, manufactured by Giba- 

Geigy. 

Beekeeping use: The use of this preparation is in the form of fumigant. 

ADI: 0.03 mg kg- 1 body weight per day or 1.8 mg per person per day. 

MRL for honey: no MRL exists for this chemical at E.U. Bromopropylate was used in crops 

such as pome fruits, stone fruits and plants of Solanacea family. The agricultural use has led 

to an establishment of MRL under the provisions of EU Regulation 396/2005. This new MRL 

corresponds to a concentration of 0.1 mg kg -1 and concerns the contamination of bee 

products through the use of agricultural pesticides. 

Bromopropylate is not toxic to bees, while its metabolite 4,4-dibromobenzolic acid is likely 

to be detected. The use of bromopropylate was particularly widespread in Central Europe. 

The observation that bromopropylate degradation is slow and, therefore very stable in 

honey and wax, forced Europeans beekeepers to start using acaricides that are more 

environment and consumer friendly. According to a survey conducted by Taccheo et al. 

(1988b), concentration of bromopropylate residues was greater in honey from an uncapped 

comb than from a capped one. Moreover, the burning of fumigant strips in an empty floor 

above the hive reduced the concentration of residues in honey (Taccheo, 1988b). The use of 

bromopropylate has stopped in many countries and no samples with residues of 

bromopropylate were found in Greek honey (Karazafiris et al., 2007). 

2.2.1.6 Malathion 

Structure: It belongs to the group of organophosphorus compounds. 

Action: malathion is broad spectrum, non-systemic insecticide-acaricide which acts through 

stomach. The way of action is similar to that of the organophosphate acaricide coumaphos. 

In addition, malathion oxidized to malaoxon, which is a substance with high toxicity to 

insects and mites. This chemical reaction does not occur in the body of mammals, limiting 

the toxicity of the acaricide for humans. 

Preparations: Malathion 

Beekeeping use: Malathion has never had approval for beekeeping use. Despite this, many 

beekeepers use it as spraying material or as powdered sugar. Due to the high bee toxicity, 

malathion requires special attention during application. A slightly increased dose can be 

fatal for the colony, especially when used as a solution. 



98 Pesticides in the Modern World - Risks and Benefits 

ADI: 0.03 mg kg- 1 body weight per day or 1.8 mg per person per day. 

MRL for honey: No MRLs have been established in honey, and therefore the threshold 
corresponding to the LOQ is 0.01 mg kg- 1 . 

Two different studies were conducted by Thrasyvoulou et al. (1988) and Balayiannis et al. 
(1989) concerning the time of degradation of malathion in honey. Both two studies proved 
that the time of degradation of malathion is three months. In a survey conducted in 50 
samples of Greek honey, 4% were found contaminated in concentrations that did not exceed 
0.005 mg kg- 1 (Thrasyvoulou et al., 1988). Futhermore, malathion was detected in 23 out of 
593 honey samples analyzed in the laboratory of Apiculture-Sericulture, Aristotle University 
of Thessaloniki during the years 2003-2006 (Karazafiris et al, 2005). In Cuba, Pelayo et al. 
(1987) detected malathion in 12 out of 110 samples. The concentration of the active substance 
did not exceed 0.02 mg kg- 1 in any case. 

2.2.1.7 Cymiazole 

Structure: It belongs to the group of iminophenyl thiazolidine. 

Action: Cymiazole is another substance, like coumaphos, that used in beekeeping and has 

systemic action. 

Preparation: The name of the commercial preparation is Apitol and is manufactured by 

Giba-Geigy. 

Beekeeping use: Cymiazole can be applied in different ways. 

ADI: 1 mg kg- 1 body weight per day or 60 mg per person per day (EMEA, 1996). 

MRL for honey: MRL that was established for cymiazole was 1 mg kg- 1 , but latest E.U. 

regulations established new MRL that corresponds to LOQ (0.01 mg kg -1 ). 

2.2.2 Antibiotic residues 

The term antibiotic originally refers to any agent with biological activity against living 
organisms; however, "antibiotic" nowadays refers to substances with antibacterial, anti- 
fungal, or anti-parasitical activity. There are currently about 250 different substances 
registered for use in medicine and veterinary medicine (Kummerer & Henninger, 2003). 
Antibiotics such as tetracycline, chloramphenicol, sulfathiazole, streptomycin, tylosin, 
erythromycin etc, are commonly used by beekeepers, in order to control European 
Foulbrood Disease (EFB), American Foulbrood Disease (AFB) and Nosemosis caused by 
Paenibacilus larvae larvae, Streptococcus pluton bacteria and fungus of the genus Nosema, 
respectively. The use of antibiotics is not allowed in beekeeping since no MRLs have been 
set for honey. Some countries, like Switzerland, UK and Belgium, have established action 
limits for antibiotics in honey, which generally lie between 0.01 to 0.05 mg kg- 1 for each 
antibiotic group. An action limit is the concentration of antibiotics in honey, above which 
the sample is considered non-compliant. The presence of antibiotic residues in honey and 
other hive products is not accepted in Europe for products imported from third countries. In 
case a product is found contaminated with antibiotics then it should be destroyed and the 
producer should be penalized. In the U.S.A., Canada and Argentina, preventive treatments 
with antibiotics are considered a routine procedure to control AFB. As a result, various 
strains of P. larvae have developed resistance to antibiotics, such as oxy tetracycline (OTC). 
Such strains have been isolated in Argentina (Alippi, 2007) as well as in many areas of the 
U.S.A. (Miyagi et al., 2000). Generally, the presence of antibiotics in the environment 



Pesticide Residues in Bee Products 99 

especially in foods may lead to the rapid emerge of resistant bacterial strains and 
consequently to the demand of new substances to replace the old. Moreover, the emergence 
of resistant bacteria involves the use of powerful antibiotics leading to serious consequences 
in the normal flora of the human body. 

2.2.2.1 Chloramphenicol 

Chloramphenicol is a potent antibiotic that has limited uses; it has been declared 
carcinogenic and causing fatal aplastic anemia, which makes it an unacceptable substance 
for use in production of food products where any residue may be found. Several reports 
document human fatalities resulting from ophthalmic preparations containing 
chloramphenicol, with exposure dozes that could be found in residues in food (Settepani, 
1984). Chloramphenicol was detected in bee products, honey and royal jelly, imported from 
China and India. In 2002, alerts appeared from the U.S., Canada, and Europe that honey 
samples from China often contained traces of the antibiotic chloramphenicol with a range of 
0.3 to 34 ug kg- 1 (LOD= ug kg- 1 ). Since China did not have stringent controls on veterinary 
use of various antibiotics, this drug had been used (along with streptomycin) by the Chinese 
beekeepers to control a bacterial epidemic that affected bee hives (Dharmananda, 2003). The 
EU, in an effort to protect consumers, banned the import of Chinese products of animal 
origin since 2004. Also in Switzerland, chloramphenicol residues detected in thirteen out of 
75 (17%) of commercially obtained honey samples, ranged between 0.4 and 6.0 ug kg- 1 
(Ortelli et al, 2004). 

2.2.2.2 Tetacyclines 

Tetracycline is used by beekeepers in order to control AFB and EFB. Normally, it degrades 
in 6-10 weeks (Matsuka & Nakamura, 1990; Gilliam et al., 1979). In some cases tetracycline 
was detected in honey, even after three years, because of the high dose used by beekeepers 
(Shakaryan & Akopyan, 1973). Acidity, viscosity and organic acids of honey contribute to 
the stability of antibiotics (Gilliam et al., 1979). The treatment of the hive with antibiotics 
results in tetracycline residues in honey and wax (Gilliam et al., 1979; Corner & Gochnauer, 
1971). In two studies of Shakaryan & Akopyan (1972 & 1973), 1.2% of the initial 
concentration of the antibiotic residues remained stable even after the heating of honey for 
three successive times in 90°C (30 minutes). Tetracyclines (tetracycline, oxy tetracycline, 
chlortetracycline, doxycycline) have been found in honey in various countries. In a study 
conducted in Greece, tetracycline residues were found in 23% of the spring floral honey 
samples tested (Karazafiris et al., 2007). In another study, out of 251 greek honey samples, 
29% were found contaminated with tetracycline residues ranged from 0.018 to 0.055 mg kg- 1 
(Saridaki-Papakonstadinou et al., 2006). 

2.2.2.3 Sulfonamides 

The sulfonamides are analogues of para-aminobenzoic acid, which include sulfapyridine, 
sulfadimidine, sulfadiazine, sulfamethoxazole, sulfadimethoxin, sulfamethopyridazine, 
sulfadoxine, sulfamethoxypyridazine, sulfadoxine and sulfamethopyrazine. They are 
suspected to cause aplastic anemia, like chloramphenicol. It is the most stable antibiotic in 
honey (Bonvehi & Pajuelo, 1983). In the past, sulfathiazole was detected regularly in honey 
produced in the European countries. Beekeepers used sulfa-drugs in order to control AFB 
and EFB and in some cases Nosemosis. 



100 Pesticides in the Modern World - Risks and Benefits 

In 2002, sulfa drugs were detected in 3 out of 91 samples of honey collected from the Belgian 
market. Moreover, 12 out of 203 honey samples collected in 2003 were contaminated by 
residues of sulfonamides (Reybroeck et al., 2004). 

2.2.2.4 Streptomycin 

The problem with streptomycin is that it may cause ototoxicity and nephrotoxicity. It is 
considered more dangerous than oxytetracycline and less hazardous than sulfathiazole 
and chloramphenicol regarding side effects. According to the Food Standards Agency of 
UK, an Indian honey was found to be contaminated by streptomycin in 2003 (Mayande, 
2007). 

2.2.2.5 Fumagillin 

This is the active ingredient of the preparation Fumidil used by beekeepers to treat 
nosemosis. It could cause teratogenesis and have genotoxic effects (Stanimirovic et al., 
2007). Nowadays, it is not permitted to use fumagillin in Europe and no MRLs have been 
established, neither for honey nor for any other products of animal origin. 

2.2.2.6 Monitoring of antibiotics in bee products 

Many other antibiotics have been used worldwide. One of these is tylosine, which got an 
approval for use in the U.S.A. in the form of preparation Tylan. Moreover, beta-lactams are 
suggested to be the ideal antibiotic group in terms of efficiency and lack of residues to the 
final product. 

Fifty chestnut, pine, linden and multifloral honey samples from Southern Marmara region of 
Turkey were analysed for erythromycin residues by Liquid Chromatography-Mass 
Spectrometry. Four of the honey samples were contaminated with erythromycin residues at 
concentrations ranging from 50 to 1776 pg kg- 1 (Gunes et al, 2008). 

A percentage of 1.7% out of 3855 honey samples of European market, which was analyzed 
for antibiotic residues, were non compliant with the EU standards. Antibiotics were 
detected in the honey samples in a range of 3-10.820 pg kg- 1 , 5-4.592 pg kg- 1 , 5-2.076 pg kg- 1 , 
0.1-169 pg kg- 1 , 0.3-24.7 pg kg- 1 , 2-18 pg kg- 1 , 1-504 pg kg- 1 for streptomycin, sulfonamides, 
tetracyclines, chloramphenicol, nitrofurans, tylosine and quinolones respectively (Diserens, 
2007). 

In the period 2000-2001, samples of honey of Belgian market were monitored for the 
presence of residues of antibiotics. Streptomycin was detected in 4 out of 248 (1.6%) samples 
that, tetracycline in 2 (2.8%) and sulfonamides in 3 (4.2%) out of 72 samples analyzed. No 
residues of p-lactams and chloramphenicol were detected. In imported honey samples, 
streptomycin was detected in 51 out of 108 samples (47.2%), tetracyclines in 29 out of 98 
samples (29.6%), sulfonamides in 31 out of 98 samples (31.6%) and chloramphenicol in 40 
out of 85 samples (47.1%). Residues of p-lactams were not detected in any sample 
(Reybroeck, 2003). 

A total of 57 samples of royal jelly were collected from beekeepers and the Chinese market. 
The royal jelly was analyzed for seven fluoroquinolones used in beekeeping (ciprofloxacin, 
norfloxacin, ofloxacin, pefloxacin, danofloxacin, enrofloxacin, and difloxacin). Ofloxacin, 
ciprofloxacin and norfloxacin residues were detected in concentrations ranging from 0.012 
to 0.056 mg kg- 1 . Difloxacin was found at a concentration of 0.047 mg kg- 1 in one sample 
(Zhou etal., 2009). 



Pesticide Residues in Bee Products 101 

2.2.3 Residues of volatile insecticides in bee products 

The greater wax moth Galleria mellonella is a serious pest of stored combs and weak colonies. 
Adult female wax moths enter hives and lay their eggs on wax combs or in small crevices 
between wooden parts of the hives not easily accessible to honey bees. After few days the 
larvae hatch and begin feeding on bees-wax, pollen, cast larval skins and other remains in 
cells. This devastating activity of wax moths leads to great financial losses every year in the 
field of beekeeping. 

Strong colonies are the best control against the wax moth in the field. In comb storage 
chests, technical, physical, biological and chemical methods have been used to control the 
pest. The most effective method to avoid the destruction of combs from wax moth is their 
continuous maintenance in temperatures of the refrigerator, or their passing from the 
freezer for a short time. Cantwell and Smith (1970) confirmed that temperature lower than - 
18 C C destroys all stages of the wax moth insect (egg, immature forms and adult). Although 
this treatment requires expensive facilities, it is successfully applied nowadays protecting 
the honeycombs from the wax moth without contaminating the beehive products. 
In addition, biological and environment-friendly control method were developed such us 
the male sterile technique with gamma-rays (Jafari et al., 2010), the trapping of moths by 
using pheromone (Flint & Merkle, 1983) and the use of the bacterium Bacillus thuringiensis 
that kills the wax moth larvae when it ingests the spores (Burges & Bailey, 1968; Burges 
1997; Charriere & Imdorf, 2004). 

Chemical methods, includes substances that are considered friendly to environment like 
methyl salicylate, clove oil, formic acid, sulphur, acetic acid, basil oil and other have been used 
(Wilson, 1965; Williams, 1980; Owayss & Abd-Elgayed, 2007). Most of these compounds are 
dangerous for bee brood and human health, while they require repeated application and may 
react and destroy the metal parts of the combs. Besides these, 1,2-dibromo-ethane (DBE), 1,4- 
dichloro-benzene (p-DCB), naphthalene had been used for many years in different countries 
even though their use causes significant contamination of bee products. 

DBE is a manufactured chemical. In nature, it is produced in small amounts in the sea water, 
where it is formed, probably by algae and kelp. It is dissolved in water and by this way it 
can stay in groundwater and in soil for a long time. In air it breaks down quickly. This 
substance has been used as a pesticide in soil, and on citrus, vegetables, and grain crops. 
EPA has banned most of these uses since 1984. The same organization has also set a limit of 
0.05 ug.crrr 3 of 1,2-dibromo-ethane in drinking water (ATSDR, 1992). 

The compound p-DCB is one of the three di-chloro-benzene isomers (1,2-DCB, 1,3-DCB and 
1,4-DCB), which is commonly used as a space deodorant i toilets and for moth control. It is a 
volatile colorless to white crystalline material with a mothball-like, penetrating odor and it 
is commercially, the most important isomer (ATSDR, 2006). 

Naphthalene is a white solid substance that evaporates easily. Its major use is in the 
manufacture of polyvinyl chloride (PVC) plastics and it is also used in moth repellents and 
toilet deodorant blocks. That use of naphthalene accounted for 73% and 60% of commercial 
demand for naphthalene in Japan and the United States, respectively in 1999, (ATSDR, 
2005). 

No MRL's in honey for the above three compounds were defined until 2005 when the 
European regulation 396/2005 EC set the limit at 10 ug kg- 1 for substances for which no 
MRL had been established. This limit for p-DCB was also the Swiss Tolerance Limit (STL) 



102 



Pesticides in the Modern World - Risks and Benefits 



and was already used as action level in Greece. ADI values for DBE, p-DCB and 
naphthalene, range according to Table 3. Besides killing the moth those chemical are 
absorbed by the wax and when bees store honey into combs, they are transferred into the 
product. Laboratory comb-melting experiment showed that p-DCB is not removed from 
wax during the comb recycling (Bogdanov et al., 2004). Residues up to 0.002 mg kg -1 may be 
detected in honey due to the use of precontaminated wax. Residues of p-DCB exceeding 0.01 
mg kg -1 indicate contamination of bee product by beekeeping practices. Countries that have 
reported problems with residues from the above volatile insecticides are Germany, 
Switzerland, Greece and Turkey (Wallner, 1992; Bogdanov et al., 2004; Tananaki et al., 2005; 
Beyoglu & Omurtag 2007). 

Wallner (1992), stated in his paper that the problem of p-DCB residues in Germany is 
serious, since 50% of the analyzed honey samples had been found contaminated from 3 to 50 
ug kg- 1 . He noticed that p-DCB is very stable in honey and it cannot evaporate from the 
sealed glass containers. Finally, he stated that beeswax works like a sponge as it has large 
capacity for fat-soluble active compounds. The more the p-DCB crystals are added to combs 
the higher is the substance stored in the wax. The evaporation of p-DCB from wax is 
impossible even after prolonged ventilation. 



Compound 



ADI (mg kg- 1 bw day- 1 ) 



US EPA 



Canadian health 



1,2-dibromoethane 

1,4-dichlorobenzene 

naphthalene 



0.009 
0.03 
0.02 



0.009 
0.11 
0.02 



Table 1. ADI of three compounds that have been used against wax moths 

Bogdanov et al. (2004) analyzed Swiss commercial honey samples during five years period 
for p-DCB residues and they found that the contaminated samples ranged from 14% to 46% 
(fig. 1). The percentage of the imported samples was lower, on average 7%. Although there 
is no MRL for p-DCB, Switzerland has established a "Swiss tolerance value" (STV) for honey 
at 10 ug kg- 1 . From the total 173 Swiss and 287 imported honey samples, 13% and 0.8% 
exceeded the STV respectively. 



SO 



40 



10 



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i Swiss 
I Imported 



1997 1998 2000 2001 2002 



Fig. 1. Residues of p-DCB in honey samples in Switzerland (Bogdanov et al., 2004) 



Pesticide Residues in Bee Products 103 

Contamination of bee products by chemicals that are used against wax moths was also 
noted in Greece by Tananaki et al. (2005). Initially a multi-method had been developed for 
the determination of DBE, p-DCB and naphthalene and then this method had been applied 
in twenty five honey samples produced in different areas of Greece. The 8% of the samples 
had detectable amounts of DBE, 92% had p-DCB and 88% had naphthalene residues. 
Concentrations of naphthalene, p-DCB and DBE that exceeded 10 ug kg- 1 were measured in 
6.7%, 32% and 8% of tested samples, respectively. 

After confirming the mass contamination, beekeepers had been informed to stop the treatment 
with those chemicals and to destroy all the combs that had been treated before. Meanwhile a 
monitoring program for the residues of volatile insecticides in Greek honey was initiated by 
laboratory of Apiculture - Sericulture of Aristotle University. A total of 1,519 samples were 
analyzed during the period 2004 - 2010 (Tananaki et al., 2006). From those, 209 samples were 
bought from Greek supermarkets (commercial) while 1,310 were collected from beekeepers or 
from their associations (bulk honey). Results of this research are indicated in Fig. 2. 
Comparing the results of eight years' monitoring of p-DCB, a considerable reduction of 
residues is observed both in commercial and bulk honey samples. During the first year the 
82.9% of commercial samples had residues more than 10 mg kg- 1 which is the established 
action limit in Greece since 2005. In the following three years this percentage decreased 
gradually and finally p-DCB wasn't detected at concentrations more than 10 mg kg- 1 in 2010. 
Similar behavior was observed for the samples collected from beekeepers. These results 
demonstrate that the Greek beekeepers' efforts to restrict the problem and to find alternative 
solutions for the control of the wax-moth (Galleria mellonela) have been accomplished. 
The great percentage of commercial samples in all years of study have either no detectable 
amounts or below 10 ug kg- 1 DBE. Only one sample was found exceeding 40 ug kg- 1 in year 
2003. This sample had 60.5 ug kg- 1 DBE, which is the maximum concentration found in 
samples bought from stores. Samples that had been collected from beekeepers had higher 
concentration of DBE than the commercial ones. This is because commercial samples 
usually are mixtures from different producers. During year 2003, a percentage of 9,9% of 
the samples exceeded the level of 10 ug kg- 1 and a maximum value of 132.5 ug kg- 1 was 
found in one of them.During the following two years this percentage decreases to 1.9% 
and 2.8% respectively, but still some beekeepers continue to use the chemical as indicated 
by the high concentration of 331.2 ug kg- 1 detected in one sample in 2004. 
Figure 2 summarizes the results of naphthalene residues in honey from the Greek market 
and from beekeepers as well. Contrary to p-DCB and DBE, naphthalene was found in more 
commercial samples than in samples from beekeepers during the first year of monitoring 
program. This could be attributed to blending of Greek commercial honeys with imported 
honey originating from countries where naphthalene is still used to control wax-moth. 
During the following years the residues in commercial samples dropped below 10 ug.kg- 1 
and very few beekeepers' samples were contaminated at higher levels. The highest 
concentration of naphthalene found in one sample was 523.6 ug.kg- 1 in 2004. 
Tananaki et al. (2006) also found differences in the level and the frequency of contamination 
among different types of honeys. Honey produced during the spring honey flow (blossom 
and fir honeys) was contaminated in a higher percentage than the honey produced later in 
the season (thymus and pine honey). Thymus and blossom honey have higher 
contamination in naphthalene than other types of honey. This might happen because both 
thymus and blossom are the types of Greek honey that are probably mixed with imported 
honey. Paleologos et al., (2006), Tsimeli et al., (2008) and Harizanis et al., (2008) have also 
analyzed samples of Greek honey with similar results. 



104 



Pesticides in the Modern World - Risks and Benefits 



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Pesticide Residues in Bee Products 105 

Residues of p-DCB were also detected in royal jelly. Tananaki et al. (2009) found that the 
concentrations of p-DCB in honey were significantly lower than in the royal jelly; in some 
cases, royal jelly had some hundred times more residues than honey from the same comb. 
The maximum concentration of p-DCB found in royal jelly was 1,520 ug kg- 1 . Bogdanov et 
al. (2004) checked the p-DCB residues in wax. They analyzed wax samples from 
manufactures during the years 1994 -1998 and 2002 and they found residues in 66% of the 
wax sample, in concentrations from 0.7 to 74.9 mg kg- 1 . The concentrations of p-DCB in new 
wax after melting of old combs were the same with those of the old combs. This indicates 
that p-DCB is not being removed from wax during the comb recycling process. 

2.3 Methods of analysis of pesticide and acaricide residues detected in bee products 

The need of monitoring residues of acaricides used by the beekeeper in conjunction with the 
need to monitor the contamination of bee products from other sources, such as pesticides 
used on crops and environmental pollutants, makes the development of appropriate 
methods of analysis obligatory. Furthermore, it is necessary to analyze products like honey 
and pollen randomly in order to find any violations of existing legislation on the part of 
producers or sellers. The suitability of each method lies in its ability to give a reliable result 
of the concentration of residues. A complete method should usually includes four sub- 
stages, which are described as: 

• Sampling 

• Sample Preservation 

• Sample Preparation 

• Analysis 

The particular physicochemical properties of each product (moisture, fat, protein content 
etc.) in conjunction with specific physicochemical properties of each substance (polarity, 
volatility, etc.) do not permit the use of one methodology for the determination of all active 
substances in all products. Various techniques have been reported in order to clean -up the 
sample and isolate the analyte. 

2.3.1 Sampling 

The first step of an analysis is the sampling. Specifically, the meaning of a sample is to take a 
part of the product, which should be as representative as possible. The way of the sampling 
varies, depending on the type of sample. In homogeneous samples such as water, the 
sampling is simple and does not require complicated procedures. On the contrary, 
heterogeneous samples such as fruits, vegetables and animal products require additional 
measures during sampling in order to reduce the uncertainty. The contribution of sampling 
to the total uncertainty is so great that in some cases approaches 40%. The EU issued a 
special directive on the sampling of food (2002/63/ EK) and requires the accurate 
implementation of the official control laboratories. 

2.3.2 Sample preservation 

The second stage of the analysis is the preservation of the sample. The common practice of 
laboratories is the storage of collected samples for a period ranging from some hours to 
years. Storage conditions must ensure the preservation of the sample during the period 
required for the analysis. Food should normally be preserved under freezing conditions, 



106 Pesticides in the Modern World - Risks and Benefits 

until the day of analysis, in order to minimize the evaporation or chemical reactivity of these 
compounds. 

2.3.3 Sample preparation and analysis 

The next step includes the preparation and the analysis of the sample. The analysis is 
performed directly, i.e. without pretreatment of the sample, where a direct measurement is 
possible (e.g. measurement of moisture in honey). In most cases, however, a preparation of 
the sample should take place before the analysis. The preparation of the sample includes the 
removal of interferences and the isolation of compounds of interest. This step is necessary in 
methods of analysis for residues of pesticides and veterinary drugs. Especially in the case of 
residue analysis, this stage is divided into separate stages that vary in terms of the number 
and type. Typically, these steps are five and consist of: 

The homogenization, which may includes a stage of subsampling. 

The extraction, including the isolation of analytes in a suitable solvent. 

The removal of a significant amount of solvent in order to make the stage of purification 

of the sample easier and faster (optional step). 

The cleaning of the sample in order to remove interferences that prevent the proper 

evaluation of chromatograms. 

The final concentration of solvent, allowing qualitative identification of analytes and the 

minimization of the quantitative limits. 
The cleaning of the sample is the most complicated stage. That is the reason why various 
techniques have been used. The main techniques used for the preparation and analysis of 
honey samples are summarized in a review of Rial-Otero et al. (2007). Techniques that have 
been used in order to achieve the determination of acaricide and pesticide residues in bee 
products are: 

• Solvent Extraction (SE). This is the first technique developed in order to detect pesticide 
residues. In this technique, the sample is dissolved in water, or mixtures of water and 
alcohols. After the dilution of the sample, an extraction with suitable organic solvents 
takes place, in order to collect the analyte and remove a large portion of co-extractives 
components. Several methods of the SE used combined with acidification of the sample 
(Waliszewski et al., 1998; Waliszewski et al., 2003; Bernal et al., 1997) or the use of 
ultrasound (Jimenez et al., 2000; Rezic et al., 2005), in order to improve the efficiency. 
Due to the use of large quantities of organic solvents, the SE is particularly aggravating 
for the environment and the health of laboratory staff. Moreover, the cost is quite high 
due to the large quantity of supplies. Finally, many hours are required for analysis of a 
sample and the automation of the process is very difficult. Despite these drawbacks, the 
SE has been used with satisfactory results in various methods of analysis of honey 
(Jimenez et al., 2002; Menkissoglu-Spiroudi et al., 2000; Taccheo et al., 1988a), royal jelly 
(Balayannis, 2001), pollen and bees (Bernal et al., 1997) for the determination of 
pesticide and acaricide residues. 

• Accelerated Solvent Extraction (ASE). This technique includes steps of extraction with 
organic solvents at predetermined conditions of pressure and temperature. In ASE, 
the extraction solvent is carried out in a special device and the extraction under 
steady environmental conditions (pressure and temperature) allows efficient and 
reproducible isolation and collection of analytes. The quantities of solvents used in 



Pesticide Residues in Bee Products 107 

this technique are very small compared to the SE and the automation of the extraction 
procedure much easier. Disadvantage of this technique is the high cost of required 
equipment. However, the small amount of solvent and the possibility of automation 
make it possible to recover the cost within a short period of time (especially for 
laboratories that analyze large numbers of samples). The ASE has been used 
successfully in many cases of food and water analysis by EPA (Chuang et al v 2001). 
There is only one study on the analysis of bee products with ASE. Results indicated 
good efficacy in the determination of acaricides in honey by the use of High 
Performance Liquid Chromatography (HPLC). The recovery rates of this method 
ranged from 58% to 103% and limits of quantification ranged from 0.01 mg kg- 1 to 0.2 
mg kg- 1 (Korta et al v 2002). ASE is likely to be referred in the literature with the 
names of PLE (Pressurized Liquid Extraction), PSE (Pressurized Solvent Extraction) 
and PFE (Pressurized Fluid Extraction). 

• Supercritical Fluid Extraction (SFE). The SFE is a technique similar to the ASE with 
similar advantages and disadvantages, while the equipment used for this technique is 
rather expensive (Mitra, 2003). The difference between the two techniques lies in the 
type of solvent, which is carbon dioxide (CO2) for the SFE. Adding a small amount (1- 
10%) of an organic solvent (such as methanol, ethanol, etc.) improves the efficiency of 
extraction of more polar compounds, which otherwise would be very small. Two types 
of supercritical fluid extraction techniques, called static and dynamic were developed. 
In the case of static SFE, the solvent enters the cell, which contains the lyophilized 
sample and remains an exact time at constant pressure and temperature conditions. 
However, in the dynamic SFE, the flow of solvent into the cell remains constant and 
stable for perfectly accurate time and at constant pressure and temperature conditions. 
The final extract is transferred to a vial containing an organic solvent. The SFE is a rapid 
technique that requires very small quantities of organic solvents and does not 
contaminate the environment significantly. Unlike ASE, there are several publications 
on the analysis of residues in honey using SFE. Rissatto et al. (2004) developed a 
method to analyze samples of honey combined SFE system and gas chromatography. 
The limit of quantification was 0.01 mg kg- 1 , while recovery rates ranged from 75% to 94 
%. In a second study conducted by Atienza et al. (1993) the average recovery rates 
ranged from 53% -94% while the RSD of the method ranged from 1.3% to 1.6%. In one 
case, this technique was used for the analysis of organophosphorus and carbamate 
insecticide residues in bees. The recovery rate exceeded 75% for all substances except 
omethoate (Jones & McCoy, 1997). 

• Gel Permeation Chromatography (GPC). This technique allows the separation of 
different components based on their size (larger particles move faster). Gels of various 
porosity and organic solvents are used in order to achieve the separation. Usually, this 
technique is used to remove lipids, proteins, polymers and other macromolecules 
contained in the sample. Especially for the pesticide analysis, the technique is suitable 
for removing high boiling point compounds, which are deposited to the inlet of gas 
chromatography. Rossi et al., (2001) have used the GPC on the analysis of residues in 
bees. The recovery was satisfactory for 25 of 29 substances analyzed (percentage 
recovery ranged from 70.9% to 106.8%). In contrast, the recovery rate for active 
substances pirimicarb, ethiofencarb, methiocarb and fenoxycarb was 38.7%, 48.6%, 
46.6% and 58.4% respectively. 



108 Pesticides in the Modern World - Risks and Benefits 

• Stir Bar Sorptive Extraction (SBSE). This extraction technique is using an appropriate 
stirring bar, which adsorbs the analyte. The bar was either eluted with suitable 
organic solvents or placed directly to the inlet of gas chromatography systems 
(Baltussen et al v 1999). Particularly, the bar is made by stainless steel coated with a 
thin layer of glass and poly-dimethyl siloxane (PDMS), which adsorbs the analyte in 
the sample (Popp et al., 2001). It is very important for the efficiency of extraction to be 
accurate in temperature and extraction time. The greater the precision, the more 
improved the repeatability of the method. In the final phase, the bar is placed in a 
special unit, which in turn is attached to the inlet. The adsorbed substances led to the 
column and detector with a carrier gas flow rate increasing with temperature. There 
is also the option for the bar to be extracted with organic solvents (e.g. acetonitrile), 
which constitute the final sample for chromatographic analysis (Sanchez-Rojas et al., 
2008). SBSE has been used on bee products with good results compared the SPME. 
Based on data given by Blasco et al. (2004), the SBSE is more efficient as a technique 
than SPME, while accuracy and repeatability are much better. More specifically, the 
limit of quantification was 0.04 mg kg- 1 for SBSE technique, while those for SPME 
techinique ranged from 0.8 mg kg -1 to 3.0 mg kg- 1 . Moreover, the recovery of SBSE 
ranged from 40% to 64%. Finally, the relative standard deviation of repeatability did 
not exceed 10% in both cases. 

• Solid Phase Micro Extraction (SPME). The SPME is a relatively modern technique, 
developed by Pawlyszin et al. (1997). The principle of this technique relies on the use 
of a fiber, which adsorbs the analyte, which then eluted to the inlet of gas 
chromatography systems. The SPME technique is suitable for the determination of 
volatile compounds in liquid or solid samples. SPME can be used in two ways. The 
first method involves an extraction by sinking the fiber into the sample solution 
directly. This is an advantage in terms of sensitivity and the number of identified 
substances. The second way relates to cases of extraction in the supernatant layer of 
sample. The advantage of this method is the higher level of purity of the final sample 
(Arthur & Pawliszyn, 1990; Louch et al., 1992; Zhang & Pawliszyn, 1993; Page & 
Lacroix, 1993). Both SPME and SBSE are based on the logic of the adsorption of 
chemicals in various absorbents, which in the first case is a fiber (SPME), and in the 
second a bar (SBSE). Another important parameter, which can greatly improve the 
results of SPME, is pH (Volante et al., 2001). The adjustment of pH by using buffers 
could improve efficiency or reduce the time of extraction. It should be noted that 
there is a variety of fibers, which differ in the type and thickness of the adsorbent 
material. The advantages of SPME include: (i) the lack of use of organic solvents, (ii) 
the purest final samples, (iii) the minimization of time, (iv) the good linearity of the 
method, (v) the non-requirement for full adsorption of the analyte & (vi) the 
relatively simple automation (Pawliszyn, 1997). The major disadvantage of SPME is 
the low efficiency for the semi-volatile or non-volatile compounds and the inability to 
repeat the analysis of a sample (same bottle). SPME was used for the detection of 
pesticide and acaricide residues in honey. More specifically, the fiber of SPME was 
immersed in an aqueous solution of honey and remained there until equilibrium of 
the analyte between the fiber and the environment was achieved. After this, the fiber 
was removed and placed in the inlet of gas chromatography in order to desorb the 



Pesticide Residues in Bee Products 109 

ingredients. The period of immersion of the fiber, as well as the temperature was 
strictly defined and determined by tests during the development of the method. The 
technique of solid phase microextraction has been applied for the determination of 
OCP, OPP, pyrethroid and acaricide residues in honey (Blasco et al v 2004; Yu et al., 
2004; Jimenez et al., 1998). In a comparative study, two different types extraction 
fibers (PDMS 7 mm, PDMS 100 mm and PA 85mm) were tested. The fiber made of 
PDMS proved significantly superior in terms of reproducibility, sensitivity, linearity 
and time of extraction obtained (Jimenez et al., 1998). 

• Matrix Solid Phase Dispersion (MSPD). The MSPD includes a stage of dilution of the 
sample in an organic solvent (e.g. methanol) and mixing a quantity of the solution with 
a sorbent, which is usually Cis or Florisil. Next phase involves addition of solvents 
(hexane, ethyl acetate, etc.), working as means of extraction and elution. After good 
homogenization in an ultrasonic bath and centrifugation, the extract is collected and 
analyzed in chromatography systems. The advantages of this technique include the 
limited use of solvents and the rapid process of the sample. Although MSPD was a 
promising technique, it is expected to be replaced by QuEChERS, which is a new 
method of analysis described in next paragraph. The MSPD is rarely used in the 
analysis of acaricide and pesticide residues in bee products. However, there are few 
studies used MSPD and gas chromatography for the detection of pesticides in honey. 
Limits of quantification in these studies were lower than 0.015 mg kg- 1 for any pesticide, 
while the recovery ranged between 60% and 113% (Albero et al., 2001; Sanchez et al, 
2002). 

• QuEChERS. The name of the technique derives from the characteristics of this 
method, which is described as Quick, Easy, Cheap, Effective, Rugged and Safe 
(Schenck & Hobbs, 2004). The QuEChERS is a new technique used for the 
determination of pesticide residues in food analysis. This technique is based on solid- 
phase dispersion extraction (Matrix Solid-Phase Dispersion). QuEChers developed 
and validated by Anastassiades (2005) and quickly began to be used by many 
laboratories. Nowadays, QuEChERS is the common sample preparation technique of 
official laboratories of European Union. This technique was developed primarily for 
the analysis of products with high water content. The addition of water to the sample 
makes possible the use of this technique for the analysis of products like honey. The 
disadvantage of this technique is the need of expensive equipment (GC/MS/MS, 
LC/MS/MS etc.), because of insufficient "cleanup step" of the sample. QuEChERS 
was used in order to detect residues of 36 pesticides in honey. Honey samples were 
extracted with acetonitrile. The extraction step was followed by the addition of acetic 
acid with the simultaneous addition of magnesium sulphate and sodium acetate. A 
mixture of primary/ secondary amine (PSA) and magnesium sulphate was added as a 
second purification step. This step was followed by a change of solvent with a 
mixture of hexane and acetone. The quantification of organophosphorus compounds 
carried out using a nitrogen phosphorus detector (NPD), while an electron capture 
detector (ECD) was used for the determination of chlorinated hydrocarbons and 
pyrethroids. Recovery experiments were made at three levels (from 0.02 mg kg- 1 to 5 
mg kg- 1 ) and the results ranged from 70% to 120%. Experimental repeatability was 
satisfactory, as the RSD ranged from 1% to 22%. Finally, the expanded uncertainty 



110 Pesticides in the Modern World - Risks and Benefits 

was relatively high (30%), but within the limit of 50% provided in pesticide residues 
analysis (Barakat, 2007). 
• Solid Phase Extraction (SPE). It is the most widely used technique of last decades, in 
the case of analysis for pesticide and veterinary drug residues. The solid phase 
extraction is the perfect choice for most researchers, since it requires a small amount 
of organic solvent (and thus is environmentally friendly), is easily automated and 
requires no expensive equipment. The disadvantages are the more expensive 
consumables (solid phase extraction microcolumns), the specialized staff, the 
differences between lots of microcolumns and the possible absorption of some 
substances on the polypropylene used in cartridges. Specifically, the sample is 
dissolved in water (Jimenez et al., 2000; Bernal et al., 1996), alcohol (Bernal et al., 
2000) or mixtures of them (Karazafiris et al., 2008; Jimenez et al., 2008), followed by 
activation of the microcolumn with the same solvent. Subsequently, the sample is 
passed through a microcolumn containing a suitable solid material, which captures 
the analyte. The bound substances are eluted with the passage of an appropriate 
organic solvent. In the case of honey acetone (Bernal et al., 1996), dichloromethane 
(Jimenez et al., 1998), ethyl acetate (Tsigouri et al., 2001), hexane (Gomis et al., 1996), 
methanol (Bernal et al., 2000), a mixture of hexane- ethyl acetate (Tsigouri et al., 2001) 
have been occasionally used. With regard to the types of substrates used occasionally, 
the reverse phase Ci8 was the most appropriate and chosen by most researchers for 
the extraction of insecticides, acaricides, herbicides, fungicides and other pesticides 
(Jimenez et al., 2000; Bernal et al., 2000; Korta et al, 2001). Also microcolumn with 
Florisil gave good results in trials for determination of pyrethroid, OCP and OPP 
residues (Jimenez et al., 1998a) and Cs in the determination of tau fluvalinate 
(Tsigouri et al., 2001). The pH adjustment proved particularly important for the good 
recovery of some active substances. For example, coumaphos is unstable in an 
alkaline environment, as opposed to amitraz, the recovery increases with increasing 
pH values up to 11 (Korta et al., 2001). A comparison of the effectiveness of SPE and 
SE in pesticide residue analysis was conducted in two publications by Bernal et al. 
(1996 & 2000). According to the results of the comparison, it should be noted that the 
recovery rate with both techniques was similar, but SPE proved superior to the purity 
of the chromatograms. The analysis of royal jelly using the technique of solid phase 
extraction was first mentioned, by Karazafiris et al., (2008b). The method proved 
efficient for the determination of acaricide and insecticide residues. 

2.3.4 Determination of analytes 

The isolation of analytes from the matrix is followed by the necessary step of separation. The 
practices used on pesticide and veterinary drug residue analysis are based on 
chromatographic methods. The choice of gas or liquid chromatography is mainly based on the 
chemical properties of analytes. The technique of gas chromatography was proved suitable for 
the determination of volatile and low molecular weight compounds, in contrast to the 
technique of liquid chromatography, which was used in less volatile and high molecular 
weight substances. The methods of analysis are classified according to the number of 
compounds detected. The two main categories are multi residue methods (MRM) and single 
residue methods (SRM). The majority of compounds identified with multi residue methods. 



Pesticide Residues in Bee Products 111 

The analyst is able to detect many different compounds by preparing and analyzing a sample 
only once. However, there are certain compounds, which can be identified individually and 
only with the use of complex techniques (e.g. amitraz in honey or pear after derivatisation). In 
recent years, due to the significant improvement of the equipment (GC-MS-MS, LC-MS-MS, 
etc.) the number of identified substances has increased considerably and many laboratories 
can identify the majority of active ingredients. The improvement of the quality and quantity of 
results issued by laboratories has been particularly important. The development of methods 
includes the use of chromatography described below. 

2.3.4.1 Gas chromatography (GC) 

The gas chromatography was used more than any other method to determine pesticide and 
acaricide residues in bee hive products. As mentioned above, this technique is mainly used 
for determination of volatile and low molecular weight compounds, but there are cases 
where higher molecular weight compounds (e.g. amitraz), analyzed by gas chromatography 
after laborious and time consuming processes (e.g. derivatization). In these cases, the 
substances were converted into more volatile compounds and then analyzed using gas 
chromatographic system. Gas chromatographic systems consist of three main sections 
outlined below: 

a. The first main section is called inlet and ensure the entrance of the sample in a gas 
chromatograph. The types of inlets used in gas chromatography is the Cool On 
Column, purged packed and split/ splitless. The type of inlet may be a problem for 
some classes of substances that are sensitive to high temperature (e.g. methamidophos 
and dichlorvos gave better results in Cool on Column inlets due to the lower 
temperature). The injection in a Cool on Column inlet takes place at low temperatures 
and benefits in repeatability and stability of analytes. The problem in this case is the 
more frequent maintenance of the column. Instead, the split/ splitless inlet advantages 
in the purity of the sample, since the majority of high molecular weight substances are 
removed by a flow of gas and do not enter the column. The result is the extension of the 
lifetime of the column and the less frequent maintenance. In return, the above 
advantages of the split/ splitless inlet may indicate the low reproducibility due to the 
removal of a quantity of analyte during cleaning. 

b. The second part consists of the oven and column at which the separation of analytes 
happens. The separation of substances achieved with the strictly programmed 
temperature and carrier gas flow within the oven and column respectively. The 
repeatability of retention time of an analyte depends on the repeatability of the above 
conditions. The column packing material is a very important factor for the separation 
and identification of various substances. Small to medium polarity columns are usually 
used for the detection of acaricides and pesticides. The use of more polar columns is 
necessary in some cases of single residue methods (e.g. determination of amitraz and its 
metabolites in honey and beeswax). Finally, a factor worth mentioning is the quality of 
the gases (carrier, auxiliary gas, etc.) that can substantially improve the sensitivity and 
lifetime of the column and the detector. 

c. The inlet and the column associated with a suitable detector. Detector achieves the 
visualization of the result. In the case of beehive products the following detectors have 
been used: 



112 Pesticides in the Modern World - Risks and Benefits 

• Electron Capture (ECD), to detect pyrethroid, organochlorine and 
organophosphorus insecticide and acaricide residues (Baltussen et al., 1999; 
Barakat et al., 2007; Jimenez et al., 1996; Jimenez et al., 1998a; Karazafiris et al., 
2008b; Menkissoglu-Spiroudi et al., 2000; Rissato et al., 2004). 

• Nitrogen-Phosphorus (NPD), to detect pyrethroid and organophosphorus 
insecticide and acaricide residues (Balayannis, 2001; Baltussen et al. 1999; Jimenez 
et al., 1998b; Menkissoglu-Spiroudi et al, 2000). 

• Flame Ionization (FID), to identify residual acaricides (Bernal et al., 2000). 

• Atomic Emission (AED), to detect acaricide residues (Jimenez et al., 1996). 

• Mass Spectrometry (MSD), to detect pyrethroid, organophosphate, carbamate and 
organochlorine insecticide or acaricide residues (Albero et al., 2004; Baltussen et al., 
1996; Bernal et al, 1996; Chauzat et al., 2006; Rissato et al., 2004). 

• Flame Photometric Detector (FPD) and Pulsed Flame Photometric Detector (PFPD), 
for detection of organophosphorus insecticide and acaricide residues (Yu et al., 
2004). 

Each chromatographic system may include components that automate the process and 
provide valuable assistance to the analyst. The most important component in optimizing the 
analytical procedure is the autosampler. The performance of a chromatographic system is 
maximizing by the use of autosampler, while it improves the reproducibility of injection 
volume and the number of samples, which can be analyzed daily. 

2.3.4.2 Liquid chromatography (LC) 

Unlike gas chromatography, which is limited to determining the most volatile compounds, 
liquid chromatography is used for the isolation of a widespread group of compounds. These 
compounds may not be sufficiently volatile or heat-resistant to analysis by gas 
chromatography. The most common types of detectors used in liquid chromatography were 
Diode Array Detectors (DAD) (Atienza et al, 1993; Blasco et al, 2004; Jones & McCoy, 1997; 
Martel & Zeggane, 2002), Ultraviolet/Visible Detectors (UVD) (Jimenez et al., 2000) and 
Fluorescence Detector (FLD) (Bernal et al., 1997). The detection technique that is gaining 
ground is mass spectrometry (MS) (Blasco et al., 2004; Chauzat et al., 2006; Fernandez et al., 
2002). In particular, the mass spectrometer with a triple quadrupole is concerned the most 
suitable detector for pesticide and veterinary drug residue analysis. The above technique 
enables determination of the majority of active substances, combined with excellent 
sensitivity (LOQ of about 0.001 mg kg- 1 for most of analyzed substances) and fewer 
requirements for the cleaning of the sample. The mass spectrometry is the ideal detector in 
conjunction with the QuEChERS method referred above. In each case, the high cost of the 
equipment and the need of qualified scientific staff should be noted. The packing material 
and the size of the column play an important role in the analysis with HPLC. The most 
widely used column is a C18 reverse phase with an internal diameter of 4.6 mm id. There 
are also columns with different packing material (C8, ODS, etc.) and columns with very 
small internal diameter (e.g. 2.1 mm id and 0.32 mm id), which help to increase the 
sensitivity and reduce the quantities of solvents used (Atienza et al., 1993). The mobile phase 
used in liquid chromatography is solvents such as water, methanol and acetonitrile or 
mixtures of them. Also, the adjustment of pH of the mobile phase plays an important role in 
the effectiveness of the method. In most cases a value of pH=9 is ideal for analysis of 
pesticide residues. 



Pesticide Residues in Bee Products 113 

2.3.4.3 Thin layer chromatography (TLC) 

This technique is used primarily for detecting drugs in biological samples. However, TLC 
was used to determine pesticide residues in food. More generally, the TLC requires sample 
extraction with a solvent mixture and separation of the components into blocks with a 
suitable coating material (e.g. Silica gel). The next step is an elution with suitable solvents. 
Special equipment is necessary in order to achieve the visualization and quantification of 
results. The TLC was used by Rezic et al. (2005) to detect residues of herbicides atrazine and 
simazine in honey. The recovery rate was estimated at 92.3% and 94.2% for atrazine and 
simazine respectively. The TLC was used in the above study in conjunction with the use of 
ultrasound during the extraction. 

2.3.4.4 Matrix effect 

A fact that has to be mentioned is that differences in the chemical synthesis of bee products 
may affect the efficiency of extraction (Blasco et al., 2004; Yu et al., 2004) and the response of 
the chromatographic systems to analytes (Volante et al., 2001; Jimenez et al., 1998; 
Karazafiris et al., 2008b). This is the reason why, solutions for calibration curve and 
recoveries should be prepared in an extract of the same sample analyzed. Specifically, the 
analyst applies the chosen technique in honey or other hive products containing no 
detectable residues of analytes. The final extract is derived from the overall process used in 
the construction of standard calibration curves. If it is not possible to find sample with no 
residues, an analyst can use an extract of the sample that gives a response 30% over the 
reference value. The response may be due to the presence of the analyte or an interference 
eluting at the same retention time. 

2.4 Methods for the determination of volatile insecticide residues in bee products 

The research on the detection of volatile insecticides residues from substances that are used 
against Galleria mellonella has been started twenty years ago. Various methods of isolation 
and analysis have been developed which are mainly based on chromatographic separation. 
Table 2 summarizes all those methods with some analytical information and the 
corresponding references. 

During the first SMPE isolation method a small amount of honey was diluted with water 
and transferred to the vials. The p-DCB molecules were collected on PDMS-fiber (5 cm, 
100 um) and the adsorption process took place for 45 min at 20-25 °C. Desorption was 
performed by raising the fibre temperature to 250 °C for 15 min and the analytes 
transferred to the GC column (DB-5ms: 30m x 0,25mm, 0,25pm). The detection was 
achieved with a MS detector at the level of 1 pg kg- 1 (Bogdanov et al., 2004). Tananaki et 
al. (2005) developed a sensitive method for the simultaneous determination of p-DCB, 
EDB and naphthalene residues in honey, using a purge and trap - gas chromatography - 
mass spectrometry system (P&T-GC-MS). In this research the analytes were extracted by 
He purging and then they absorbed onto the Tenax resin. With thermal desorption the 
isolated compounds were transferred to the GC - MS system. Separation was performed 
on a fused silica capillary column (30mx0.25mm I.D., 0.25 pm film thickness). The limits 
of detection were found to be 0.8, 0.15 and 0.05 pg kg -1 honey, while the limits of 
quantification were 2.4, 0.5 and 0.125 pg kg -1 for EDB, p-DCB and naphthalene 
respectively. 



114 



Pesticides in the Modern World - Risks and Benefits 




Table 2. Methods for the determination of volatile insecticides residues 



Pesticide Residues in Bee Products 115 

The acid-induced liquid-liquid phase separation of anionic surfactants in aqueous 
solutions and its applicability to cloud point extraction methodology were applied as a 
tool for the extraction of 1,4- dichlorobenzene (p-DCB) from aqueous honey samples. The 
analyte is extracted into the micelles of sodium dodecane sulfonate. For the separation of 
p-DCB a high-performance liquid chromatographic equipped with a UV detector system 
(225 nm) was used (Paleologos, et al. 2006). Dichlorobenzene and naphthalene residues in 
honey were investigated by solid-phase microextraction (SPME) coupled to gas- 
chromatographic/mass spectrometry from Harizanis et. al (2008). The equilibration time 
and the sampling time for the extraction of the analytes by the fibre was 30 min and 60 min 
respectively, while the honey solution was kept at 60 °C The LOD and LOQ for the p- 
dichlorobenzene was 1 ug kg -1 and 5 ug kg -1 - while for naphthalene 0.1 ug kg -1 and 1 ug kg -1 
respectively. 

Tsimeli et al. (2008) developed a method for the determination of DBE, p-DCB and 
naphthalene based on SMPE extraction. Commercially available 100 um film thickness poly- 
dimethylsiloxane (PDMS) fiber was employed for the extraction. The fibre was exposed to 
the headspace above the sample for 30 min, while the sample was kept at 40±2 °C and 
stirred at 900 rpm. The separation and detection are carried out using gas chromatography - 
mass spectrometry (GC/MS) in selected ion monitoring mode (SIM). 

For the determination of naphthalene in honey, a high-performance liquid chromatography 
with a diode array detector method was also used. The compound was detected at 220 nm 
and the limit of detection and the limit of quantification were 0.023 ug g- 1 and 0.078 ug g- 1 
respectively (Beyoglu & Omurtag 2007). 

The p-DCB molecules were extracted from the bee wax with ethanol and the sample clean 
up was accomplished by solid-phase extraction (Cis columns), while the determination was 
achieved by capillary GC and FID detector. The detection limits of the method were 0.7 mg 
kg- 1 while average recovery was 74.8±5.5% (Bogdanov et al., 1998; 2004). For the isolation of 
p-DCB from the royal jelly a Purge and Trap system was used (Tananaki et al., 2009). The 
molecules of this compound extracted from the aqua royal jelly solution by He purging at 
40 ml min- 1 for 40 min keeping the sample temperature at 40 °C and were absorbed on 
Tenax resin. For the separation a fused silica capillary column (HP-5MS) has been used, 
while the detection was achieved using a mass spectrometer detector. The LOD and LOQ of 
the method was 0.3 ug kg- 1 and 0.9 ug kg- 1 respectively. 

3. Conclusion 

To maximize the production of agricultural products, extended amount of insecticides, 
herbicides, fungicides and bactericides are used which eventually lead to contamination of 
water, soil, crops, animals, even humans. Many environmental studies are concerned with 
the bioavailability of these pollutants and their subsequent introduction into food chain. 
Pesticides, Persistent Organic Pollutants (OCs, PCBs, PBBs), toxins and heavy metals have 
been investigated worldwide as substances that contaminate man's food. Chemicals 
contaminate hive products like honey, wax, pollen, propolis and royal jelly, while residues 
may exceed the established MRLs, either because of the improper use of the products or the 
utilization of unauthorized products by the beekeepers. 

Honeybees forage over a circular area, with radius more than 6 Km, visiting numerous plant 
species and various sources of water and are notorious for collecting materials contaminated 
with chemicals and bringing them back to the hive. In anyway, pollutants may reach the 



116 Pesticides in the Modern World - Risks and Benefits 

hive products and this justifies consumers' concern on this subject. According to research 
studies, the risk for bee products contamination with pesticides from the environment is 
low. Concentrations of pesticide residues detected are below LOQs in most studies, while 
there are only few cases that high concentrations of pesticides were detected in bee 
products. Moreover, antibiotics used as plant protection products can contaminate bee 
products, but the concentrations detected are low. 

Besides the above indirect method of pesticides transferring into bee's nest, the bigger risk 
for bee products contamination is the beekeeping practices. Diseases attack bee colonies and 
the beekeepers use acaricides, antibiotics, fungicide and other chemicals inside the hive to 
control them. 

Antibiotics played an important role as effective chemotherapeutics for bee diseases and 
have been used until recently. However, the use of antibiotics against any bee disease is not 
permitted in Europe anymore, because pharmaceutical companies did not apply and 
support the experimental data for MRLs in bee products as required by the European 
Medicinal Evaluation Agency (EMEA). Despite of this forbiddance, monitoring results 
indicate that antibiotic residues are still present in European honeys, but the detection 
frequency is decreasing after the European ban. Antibiotic residues are usually detected in 
honey and royal jelly, while the concentrations are very low comparing to other products 
such as milk, eggs etc. 

Another source of contamination that is caused by beekeepers is the chemicals that they use 
against Varroatosis, a disease caused by the parasitic mite Varroa destructor Anderson and 
Trueman. Varroas' presence causes many troubles to the bees including appearance of other 
diseases like sacbrood, American and European foulbrood. If it is left untreated it could 
destroy the whole colony within 2-3 years period. Varroatosis is actually the only disease of 
bees against which the use of pharmaceutical products within the hive is permitted. In 
European countries, there are authorized chemicals that can be used and limits (MRLs) that 
should not be exceeded. The contamination of bee products by acaricides can be minimized 
by careful use of the chemotherapeutic products. As far as we know the percentage of honey 
samples containing residues exceeding MRLs is low. A major problem could be the use of 
unauthorized products in order to control Varroatosis. 

Additional problem for the quality of bee products was the volatile insecticides and other 
chemicals that beekeepers used in storehouses to protect bee combs from the larvae of the 
insect Galleria mellonella (wax moth). This insect attacks the honeycombs during storage and 
can even damage the wooden frames in which they hang. The devastating activity of these 
insects is known to beekeepers all over the world. To save the combs, beekeepers use several 
chemical fumigants that are incorporated into wax and from there they are readily 
translocated into bee products. Some of those like PDCB, DBE and naphthalene pose a 
potential health hazard. Although beekeepers stopped using these compounds, residues 
were still in old combs for many years and readily transferred into honey. Volatile 
insecticide residues detected in bee products are below LOQ in countries like Greece, which 
had a major contamination problem the previous decade. 

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Pesticide Residues in Bee Products 125 

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Part 2 
Environmental Impacts of Pesticides 



Ecological Effects of Pesticides 

Zacharia, James Tano 

University ofDar es Salaam, 
Dares Salaam University College of Education, 

Tanzania 



1. Introduction 



1.1 Definition of ecology 

At a community level, ecology can be defined as complex interactions that exist among 
interdependent organisms that cohabitate the same geographical area and with their 
environment (Johnson and Strinchcombe, 2007). At individual level, it entails the 
relationships that exist between that particular individual with numerous physical and 
biological factors. The physical environment along with organisms (biota) inhabiting a 
particular space make up an ecosystem. Some typical examples of ecosystems include a farm 
pond, a mountain meadow and rain forest. In a natural environment, an ecosystem follows 
a certain sequence of processes and events through the days, seasons and years. The 
processes include not only the birth, growth, reproduction and death of biota in that 
particular ecosystem, but also the interactions between species and physical characteristics 
of the geological environment. From these processes and interactions, the ecosystem gains a 
recognizable structure and function, and matter and energy are exchanged and cycled 
through the ecosystem. Over time, better adapted species come to dominate; entirely new 
species may change, perhaps in a new or altered ecosystem. 

1.2 The organisation in ecosystems 

The basic level of ecological organisation is with the individual such as a single plant, insect 
or bird. The definition of ecology is based on the interactions of organisms with their 
environment. In the case of an individual, it would entail the relationships between that 
individual and numerous physical (rain, sun, wind, temperature, nutrients, etc.) and 
biological (other plants, insects, diseases, animals, etc.) factors. The next level of 
organization is the population. Populations are no more than a collection of individuals of 
the same species within an area or region. We can see populations of humans, birch trees, or 
sunfish in a pond. Population ecology is concerned with the interaction of the individuals 
with each other and with their environment. 

The next, more complex, level of organization is the community. Communities are made up 
of different populations of interacting plants, animals, and microorganisms also within 
some defined geographic area. Different populations within a community interact more 
among themselves than with populations of the same species in other communities, 
therefore, there are often genetic differences between members of two different 
communities. The populations in a community have evolved together, so that members of 
that community provide resources (nutrition, shelter) for each other. 



130 Pesticides in the Modern World - Risks and Benefits 

The next level of organization is the ecosystem. An ecosystem consists of different 
communities of organisms associated within a physically defined space. For example, a forest 
ecosystem consists of animal and plant communities in the soil, forest floor, and forest canopy, 
along the stream bank and bottom, and in the stream. A stream bottom community, for 
example, will have various fungi and bacteria living on dead leaves and animal wastes, 
protozoans and microscopic invertebrates feeding on these microbes, and larger invertebrates 
(worms, crayfish) and vertebrates (turtles, catfish). Each community functions somewhat 
separately, but are also linked to others by the forest, rainfall, and other interactions. For 
example, the stream community is heavily dependent upon leaves produced in the 
surrounding trees falling into the stream, feeding the microbes and other invertebrates. 
Terrestrial ecosystems can be grouped into units of similar nature, termed biomes (such as a 
"deciduous forest," "grassland," "coniferous forest," etc.), or into a geographic unit, termed 
landscapes, containing several different types of ecosystems. Aquatic ecosystems are 
commonly categorized on the basis of whether the water is moving (streams, river basins) or 
still (ponds, lakes, large lakes) and whether the water is fresh, salty (oceans), or brackish 
(estuaries). Landscapes and biomes (and large lakes, river basins, and oceans) are subject to 
global threats of pollution (acid deposition, stratospheric ozone depletion, air pollution, the 
greenhouse effect) and human activities (soil erosion, deforestation, pesticides use). 

1.3 Why pesticides are unique among environmental contaminants 

Pesticides released into the environment may have several adverse ecological effects ranging 
from long-term effects to short-lived changes in the normal functioning of an ecosystem. 
Despite the good results of using pesticides in agriculture and public health, their use is 
usually accompanied with deleterious environmental and public health effects. Pesticides 
hold a unique position among environmental contaminants due to their high biological 
toxicity (acute and chronic). Pesticides by definition are toxic chemical agents. A pesticide is 
usually capable of harming all forms of life other than the targeted pest species. On account 
of this behavior then, they can best be described as biocides (capable of killing all forms of 
life). Although some pesticides are described to be selective in their mode of action, their 
range of selectivity is only limited to the test animals. 

1.4 The vast potentials of pesticides distribution and fate in the environment 

The term chemodynamics of pesticides refers to the study of the movement and 
transformation of pesticides as well as their fate in various compartments of the 
environment. The environment can be divided into four major compartments, namely; air, 
water, soil and biota (Fig.2. 1). 

The widespread use and disposal of pesticides by farmers, institutions and the general 
public provide many possible sources of pesticides in the environment. Pesticides once 
released into the environment may have many different fates. Pesticides that are sprayed 
can move through the air and may eventually end up in other parts of the environment, 
such as in soil or water. Pesticides that are applied directly to the soil may be washed off the 
soil into nearby bodies of surface water or may percolate through the soil to lower soil layers 
and groundwater (Harrison, 1990). This incomplete list of possibilities suggests that the 
movement of pesticides in the environment is very complex with transfers occurring 
continually among different environmental compartments. In some cases, these exchanges 
occur not only between areas that are close together (such as a local pond receiving some of 
the herbicides applied on adjacent land) but also may involve transportation of pesticides 



Ecological Effects of Pesticides 



131 



over long distances. The worldwide distribution of DDT and the presence of pesticides in 
bodies of water such as the Great Lakes far from their primary use areas are good examples 
of the vast potential of such movement. 

*- transfer czj> aoVection 

4- 



\ 




deposition 
dry/wEt 



precipitation 



runoff 





infiltration 



groundwater 



Fig. 2.1 Distribution of pesticides in different environmental compartments 

In this chapter, a closer and detailed look on the major ecological effects of pesticides are 
described based on contemporary accumulated knowledge on the behavior of pesticides and 
the damage they cause to the ecosystem and the environment at large as a result of excessive 
use and/or injudicious use of pesticides. The effects may range from minor deviation on the 
normal functioning of the ecosystem to the loss of species diversity in the ecosystem. Since 
organisms in the ecosystem live in a complex interdependent association with each other, the 
loss one key species may result in the collapse of the particular ecosystem. These effects are an 
important reason for the current strict regulations on the judicial use of pesticides. 



2. Ecological effects of pesticides 

The primary objective of using pesticides in the fields and the environment in general is to 
achieve a control of crop pests and disease vectors. This has been a deliberate human effort 
in a search for increasing agricultural yields and improving public health (Helweg, 2003). 
Pesticides applied to the environment have shown to have long term residual effects while 
others have shown to have acute fatal effects when not properly handled. Organochlorine 
pesticides for example have shown to be persistent in the environment, the result of which 
find their way to contaminate ground water, surface water, food products, air, soil and may 
affect human being through direct contact. Pesticides exposure to humans have been well 
documented to be the route cause of some diseases such as cancer, respiratory diseases, skin 
diseases, endocrine disruption, and reproduction disorders. It is this aspect of pesticide in 
the environment that has raised concern among environmental scientists to study their 
behaviour in the environment and then come out with a sound alternative so as to rescue 
the human population from their adverse effects. 

Fifty years (half a century) after Rachel Carson's eloquent warning to the world about the 
devastating effect pesticides have on birds and beneficial insects, pesticides continue to be 



132 Pesticides in the Modern World - Risks and Benefits 

a pervasive and insidious threat to the world's ecosystems. A massive chemical assult on 
our environment is launched each year. This poisonous barrage aggravates other 
pressures on our ecosystems such as expanding suburbarn development and dammed 
rivers, threatening the survival of many birds, fish, insects, and small aquatic organisms 
that form the basis of the food web. More generally, pesticides reduce species diversity in 
the animal kingdom and contribute to population decline in animals and plants by 
destroying habitats, reducing food supplies and impairing reproduction (Kegley, et al, 
1999). 

2.1 Loss of species diversity among the food chains and food webs 

Organisms in ecosystems exist in complex interdependent associations such that losses of 
one keystone species as a result of pesticides (or other causes) can have far reaching and 
unpredictable effects. A keystone species is a species that is disproportionately connected to 
more species in the food-web. The many connections that a keystone species holds means 
that it maintains the organization and structure of entire communities. The loss of a 
keystone species results in a range of dramatic cascading effects that alters trophic 
dynamics, other food-web connections and can cause the extinction of other species in the 
community. Sea otters (Enhydra lutris) for example, are known to be keystone species in 
marine ecosystems that limits the density of sea urchins (Mills, et al, 1993). 
A pesticide may eliminate a species essential to the functioning of the entire community, or 
it may promote the dominance of undesired species or it may simply decrease the number 
and variety of species present in the community. This may disrupt the dynamics of the food 
webs in the community by breaking the existing dietary linkages between species. The 
literature on pest control lists many examples of new pest species that have developed when 
their natural enemies are killed by pesticides. This has created a further dependence on 
pesticides. Finally, the effects of pesticides on the biodiversity of plants and animals in 
agricultural landscapes, whether caused directly or indirectly by pesticides, constitute a 
major adverse environmental impact of pesticides. 

2.2 Effects involving pollinators 

Some natural pollinators, such as honeybees and butterflies, are very sensitive to 
pesticides. Pesticides can kill bees and are strongly implicated in pollinator decline, the 
loss of species that pollinate plants, including through the mechanism of Colony Collapse 
Disorder (Hackenberg, 2007), in which worker bees from a beehive or Western honey bee 
colony abruptly disappear. Application of pesticides to crops that are in bloom can kill 
honeybees, which act as pollinators. The USDA and USFWS estimate that US farmers lose 
at least $200 million a year from reduced crop pollination because pesticides applied to 
fields eliminate about a fifth of honeybee colonies in the US and harm an additional 15% 
(Miller, 2004). 

Since these are important pollinators of both crops and native plants, reduced number of 
natural pollinators can therefore result into reduced seed and fruit production. This is both 
an ecological effect as well as economical effect. Bees are extremely important in the 
pollination of crops and wild plants, and although pesticides are screened for toxicity to 
bees, and the use of pesticides toxic to bees is permitted only under stringent conditions, 
many bees are killed by pesticides, resulting in the considerably reduced yield of crops 
dependent on bee pollination. 



Ecological Effects of Pesticides 



133 




Fig. 2.2 A butterfly and bee as representative natural pollinating agents for plants 



2.3 Effects on nutrient cycling in ecosystem 

A large proportional of pesticides used in the environment ultimately reaches the soil where 
soil building processes and the cycling of nutrients back into living plants is accomplished. 
Pesticides can affect the soil organisms involved in these processes directly or indirectly 
hence interfering with the natural nutrient cycling in the ecosystem. 




j/\^ Atmospheric 
deposition 




Ammonia 
volab' zation 

Fertilizer factory ^> '^ustry__ J Combustion 



Groundwater' 1 — •**> 
Fig. 2.3 Nutrient cycling in ecosystem 



134 Pesticides in the Modern World - Risks and Benefits 

Nitrogen fixation, which is required for the growth of higher plants, is hindered by 
pesticides in soil. The insecticides DDT, methyl parathion, and especially 
pentachlorophenol have been shown to interfere with legume-rhizobium chemical 
signaling. Reduction of this symbiotic chemical signaling results in reduced nitrogen 
fixation and thus reduced crop yields (Rockets, 2007). Root nodule formation in these 
plants saves the world economy $10 billion in synthetic nitrogen fertilizer every year (Fox, 
2007). When the natural nutrient cycling (figure 2.3) in the ecosystem is interfered in any 
way by pesticides or other sources of pollution, it will lead to decline in soil fertility and 
soil productivity. 

2.4 Effects on soil erosion, structure and fertility 

Many of the chemicals used in pesticides are persistent soil contaminants, whose impact 
may endure for decades and adversely affect soil conservation. The use of pesticides 
decreases the general biodiversity in the soil. Not using the chemicals results in higher soil 
quality (Johnson, 1986), with the additional effect that more organic matter in the soil allows 
for higher water retention. This helps increase yields for farms in drought years, when 
organic farms have had yields 20-40% higher than their conventional counterparts. A 
smaller content of organic matter in the soil increases the amount of pesticide that will leave 
the area of application, because organic matter binds to and helps break down pesticides 
(Lotter, et al, 2003). 

Herbicides for example can reduce vegetative cover of the ground, thus promoting soil 
erosion via runoff and wind. Soil erosion deforms the soil structure and therefore creates an 
imbalance in soil fertility. A bare land with poor soil structure and poor soil fertility cannot 
support the growth of plants on it. Ecologically this land cannot support other forms of life 
in it hence may lead to the collapse of the particular ecosystem. 

2.5 Effects on water quality 

Pesticides applied in the environment can find their way into water bodies either from the 
air or by runoff or by percolation to groundwater. There are four major routes through 
which pesticides can reach the water bodies: it may drift outside of the intended area 
when it is sprayed, it may percolate, or leach, through the soil, it may be carried to the 
water as runoff, or it may be spilled, for example accidentally or through negligence. They 
may also be carried to water by eroding soil. Factors that affect a pesticide's ability to 
contaminate water include its water solubility, the distance from an application site to a 
water body, weather, soil type, presence of a growing crop, and the method used to apply 
the chemical. Once pesticides enter water bodies they have a potential to cause harmful 
effects on human health, aquatic organisms and can cause disruptions of the aquatic 
ecosystems. This may result into a loss in fish production in streams and large water 
bodies especially where fishing is one among the major economic activities of a particular 
community. 

In the United States for example, pesticides were found to pollute every stream and over 
90% of wells sampled in a study by the US Geological Survey (Gillion, et al, 2007). Pesticide 
residues have also been found in rain and groundwater. Studies by the UK government 
showed that pesticide concentrations exceeded those allowable for drinking water in some 
samples of river water and groundwater (Bingham, 2007). 



Ecological Effects of Pesticides 



135 




Chemical < Adsorption/ ^ , Biological 

Degradation >. Desorption ^. Degradation 




Fig. 2.4 Pesticieds pathways in contaminating water bodies (Heather, et al, 1997) 



2.6 Effects on birds 

Pesticides have had some of their most striking effects on birds, particularly those in the 
higher trophic levels of food chains, such as bald eagles, hawks, and owls. These birds are 
often rare, endangered, and susceptible to pesticide residues such as those occurring from 
the bioconcentration of organochlorine insecticides through terrestrial food chains. 
Pesticides may kill grain- and plant-feeding birds, and the elimination of many rare species 
of ducks and geese has been reported. Populations of insect-eating birds such as partridges, 
grouse, and pheasants have decreased due to the loss of their insect food in agricultural 
fields through the use of insecticides. The loss of even a few individuals from rare, 
endangered or threatened species pushes the entire species close to extinction. Some 
pertinent examples associated with birds' kills as a result of pesticides include the 
insecticides diazinon and carbofuran which are well document as causing bird kills in many 
parts of the world (Kegley et al, 1999). Organochlorine insecticides such as DDT are also well 



136 



Pesticides in the Modern World - Risks and Benefits 



documented to have continued impairing avian reproduction even after years of banned 
use. Most bird kills go undocumented, with reported kills representing only a small fraction 
of actual bird mortality due to pesticides. 

Birds exposed to sublethal doses of pesticides are also afflicted with chronic symptoms that 
affect their behaviour, reproduction, and nervous system. Weight loss, increased 
susceptibility to predation, decreased disease resistance, lack of interest in mating and 
defending territory, and abandoning of nestlings have been observed as side effects of 
pesticides exposure. 




Fig. 2.5 A bird that died as a result of pesticides use (U.S. EPA) 



2.7 Effects on fish and other aquatic organisms 

A major environmental impact has been the widespread mortality of fish and marine 
invertebrates due to the contamination of aquatic systems by pesticides. This has resulted 
from the agricultural contamination of waterways through fallout, drainage, or runoff 
erosion, and from the discharge of industrial effluents containing pesticides into waterways. 
Historically, most of the fish in Europe's Rhine River were killed by the discharge of 
pesticides, and at one time fish populations in the Great Lakes in USA became very low due 
to pesticide contamination. Additionally, many of the organisms that provide food for fish 
are extremely susceptible to pesticides, so the indirect effects of pesticides on the fish food 
supply may have an even greater effect on fish populations. Some pesticides, such as 
pyrethroid insecticides, are extremely toxic to most aquatic organisms. It is evident that 
pesticides cause major losses in global fish production. Furthermore, recent laboratory 
studies of endosulfan and fenitrothion in the tilapia species from Lake Victoria in Tanzania 
indicated a high capacity of the species to absorb the two pesticides from water with rapid 
distribution in the organs each with a bioaccumulation factor of 33 and 346 L/kg fresh 
weight respectively (Henry, 2003). 

Multiple pesticides contamination are very common in water and sediments, frequently at 
concentrations exceeding the lethal limits for many species of zooplankton, small species of 
animals eaten by fish. Because of the significant high water solubility of the insecticides 
diazinon and chlorpyrifos and the herbicides simazine, diron, and EPTC are found most 
commonly in water bodies and have been associated with fish kills and decline in 
zooplankton population in aquatic environment. 



Ecological Effects of Pesticides 



137 




Fig. 2.6. Spraying an aquatic herbicide 



2.8 Effects on frogs and other aquatic amphibians 

Atrazine being one of the world's most used pesticide has recently reported by laboratory 
studies to have a effect on changing male frogs (African clawed frog; Xenopus laevis). Adult 
frogs exposed to atrazine turn female one in ten (10%). These male frogs are missing 




Fig. 2.7 Kihansi spray toads from Kihansi Gorge in Tanzania 



138 



Pesticides in the Modern World - Risks and Benefits 



testesterone and all things controlled by testesterone including sperm production. So their 
fertility is as low as 10 percent when treated in isolation, but when treated with normal 
males, they stand a zero chance of reproducing. Although 10 percent of these mutant 
females can successful mate with male frogs, their offspring are all male because they are 
genetically male frogs. The ultmate effect of this is that the sex ratios of frogs is badly 
skewed and this is very dangerous for the survival of that species (Hayes et al, 2010).Kihansi 
spray toads is one among the world's rarest amphibian species that was close to extinction 
from their natural environment in Tanzania. The species was first discovered in 1996 during 
an environment impact study for a large new hydroelectic dam in Udzungwa mountains in 
Southern Tanzania. The toads lived exclusively in a five acre zone under spray of a waterfall 
from Udzungwa mountains, hence the name Kihansi spray toads. Among other reasons that 
contributed to the decline is the use of pesticides in the environment. To rescue this rare 
species of toads, a colony of them was taken to Bronx zoo and Toledo zoo in USA where 
they were reared and bread in laboratories for 10 years. 



2.9 Pesticides disrupt the natural balance between pest and predator insects 

Broad spectrum pesticides such as organochlorine, organophosphorus and carbamate 
insecticides destroy both pest and beneficial organisms indiscriminately, thus upsetting the 
natural balance between pests and predator insects. Beneficial organisms serve many 
valuable functions in an agricultural ecosystem including pollination, soil aeration, nutrient 
cycling, and natural pest control through pest-predator relationship. Application of 
insecticides indiscriminately kills both pests and beneficial organisms. Pest populations 
often recover rapidly because of their lager numbers and ability to develop resistance, but 
beneficial organisms do not, resulting in a resurgence of the target pest as well as secondary 
pests that reproduce rapidly without natural predator to check down their numbers. This 
prompts an escalation in the use of more pesticides by the farmers in an attempt to control 
them and boost their harvest. 




Fig. 2.8 Aerial spraying of pesticides onto the crops using an aircraft 



Ecological Effects of Pesticides 1 39 

2.10 Pesticides cause pest rebound and secondary pest outbreaks 

Non-target organisms, organisms that the pesticides are not intended to be killed, can be 
severely affected by the use of pesticides. In some cases, where a pest insect has some 
controls from a beneficial predator or parasite, an insecticide application can kill both pest 
and beneficial populations. A study comparing biological pest control and use of pyrethroid 
insecticide for diamondback moths, a major cabbage family insect pest, showed that, the 
insecticide application created a rebounded pest population due to loss of insect predators, 
whereas the biological control did not show the same effect (Muckenfuss, et al 1990). 
Likewise, pesticides sprayed in an effort to control adult mosquitoes, may temporarily 
depress mosquito populations, however they may result in a larger population in the long 
run by damaging the natural controlling factors. This phenomenon, wherein the population 
of a pest species rebounds to equal or greater numbers than it had before pesticide use, is 
called pest resurgence and can be linked to elimination of predators and other natural 
enemies of the pest (Daly, et at, 1998). 

The loss of predator species can also lead to a related phenomenon called secondary pest 
outbreaks, an increase in problems from species which were not originally very damaging 
pests due to loss of their predators or parasites (Daly, et at, 1998). An estimated one-third of 
the 300 most damaging insects in the US were originally secondary pests and only became a 
major problem after the use of pesticides (Miller, 2004). In both pest resurgence and 
secondary pest outbreaks, the natural enemies have been found to be more susceptible to 
the pesticides than the pests themselves, in some cases causing the pest population to be 
higher than it was before the use of pesticide. 

2.11 Effects on human beings 

Pesticides can enter the human body through inhalation of aerosols, dust and vapor that 
contain pesticides; through oral exposure by consuming contaminated food and water; and 
through dermal exposure by direct contact of pesticides with skin (Sacramento, 2008). 
Pesticides are sprayed onto food, especially fruits and vegetables, they secrete into soils and 
groundwater which can end up in drinking water, and pesticide spray can drift and pollute 
the air. 

The effects of pesticides on human health are more harmful based on the toxicity of the 
chemical and the length and magnitude of exposure (Lorenz, 2009). Farm workers and their 
families experience the greatest exposure to agricultural pesticides through direct contact with 
the chemicals. But every human contains a percentage of pesticides found in fat samples in 
their body. Children are most susceptible and sensitive to pesticides due to their small size and 
underdevelopment. The chemicals can bioaccumulate in the body over time. Exposure to 
pesticides can range from mild skin irritation to birth defects, tumors, genetic changes, blood 
and nerve disorders, endocrine disruption, and even coma or death (Miller, 2004). 

2.12 Pesticides may cause pest resistance 

Pests may evolve to become resistant to pesticides as a result of continued use of pesticides in a 
particular environment. Many pests will initially be very susceptible to pesticides, but some 
with slight variations in their genetic makeup they become resistant and therefore survive to 
reproduce. Through natural selection, the pests may eventually become very resistant to the 
pesticide. Pest resistance to a pesticide is commonly managed through pesticide rotation, 
which involves alternating among pesticide classes with different modes of action to delay the 



140 



Pesticides in the Modern World - Risks and Benefits 



onset of or mitigate existing pest resistance. Tank mixing pesticides is the combination of two 
or more pesticides with different modes of action in order to improve individual pesticide 
application results and delay the onset of or mitigate existing pest resistance. 



Health effects of pollution 



Air pollution 





Water pollution 



i tatigue m 



dar 
Lead 



Nerve Particulate matter 
damage zone 



Volatile 
organic 
compounds 





Bacteria 

Parasites 

Chemicals 



Soil 
contamination 



v as cut. 
////less 



G astro enteritis 
Cancer risk Pesticides 




* ; r-t--r-t--i 



w* 



**** 



Nausea 
Skin irritation 



#t< 



Fig. 2.9 Impacts of pesticides on human health 

3. Summary and recommendations 

3.1 Summary 

In summary, the adverse ecological effects from pesticides occur at all levels of biological 
organization. The effects can be global or local, temporary or permanent, or short-lived (acute) 
or long-term (chronic). The most serious effects involve loss in production, changes in growth, 
development and/ or behavior, altered diversity or community structure, changes in system 
processes (such as nutrient cycling), and losses of valuable species. These ecological losses in 
turn may be economically or socially important. Hence, ecological effects are of serious 
concern in regulating pesticides use and a variety of tests have been devised to help evaluate 
the potential for adverse ecological effects of pesticides. Developing an understanding of how 
these tests and other information can be used to prevent environmental problems caused by 
pesticides is the basis for ecological risk assessment research. 



3.2 Recommendations 

Pesticides destroy the delicate balance between species that characterize the functioning 
ecosystem. With pesticides now being found routinely in drinking water, on food and in the 



Ecological Effects of Pesticides 141 

air, we are all taking part in an experiment in pesticide exposure on a global scale, but 
without the benefit of an exposed control group for comparison. For that matter we are 
likely not be able to quantify the exact risk of these exposures. Because we cannot know for 
certain the consequences of the expanding pesticides use, the rational and most protective 
course of action is to take a precaution approach phasing out the use of the most dangerous 
pesticides, reducing our reliance on toxic chemicals for pest control and promoting 
ecologically based pest management. 

The adverse effects of pesticides on humans and wildlife have resulted in research into ways 
of reducing pesticide use. The most important of these is the concept of integrated pest 
management (IPM), first introduced in 1959. This combines minimal use of the least harmful 
pesticides, integrated with biological and cultural methods of minimizing pest losses. It is 
linked with using pesticides only when threshold levels of pest attacks have been identified. 
There is also a move toward sustainable agriculture which aims to minimize use of 
pesticides and fertilizers based on a systems approach. 

There has been a growing concern recently on the promotion of organic farming which 
emphasize on techniques such as crop rotation, green manure, compost and biological 
methods of pest control to maintain soil productivity. Organic farming strictly excludes the 
use of manufactured fertilizers, pesticides, plant growth regulators, livestock antibiotics, 
food additives, and genetically modified organisms. Organic foods resulting from organic 
farming are deemed free from pesticides and hence providing an alternative source of 
quality and safe food in the future. By promoting the use of organic foods means will push 
the farmers to opt for organic farming. Market forces are a powerful incentive to encourage 
famers to go organic. 

Pesticides manufacturers should conduct long-term studies on ecosystem-wide impacts to 
demonstrate that a pesticide has no adverse effects before allowing it to be registered for use 
in the environment. The fact that present regulations view a pesticide as innocent until 
proved guilty is detrimental to the environment health. It is critical to know more about the 
long-term ecological effects of a pesticide before it is released to the environment. Using a 
combination of prior gained field experience with the existing pesticides and applying 
fundamental chemodynamic principles to newly developed compounds, we can now 
predict with some degree of accuracy the fate of new chemicals before they are even used in 
the environment. 

4. References 

Johnson, M. T.; Strinchcombe, J. R. (2007). "An emerging synthesis between community 

ecology and evolutionary biology". Trends in Ecology and Evolution 22 (5): 250-7 
Harrison, S. A. (1990). The Fate of Pesticides in the Environment, Agrochemical Fact Sheet # 

8, Penn, USA 
Helweg, C. et al (2003). Fate of pesticides in surface waters, Laboratory and Field 

Experiments; Ministry of Environment, Danish Environmental Protection Agency,. 

Pesticides Research No. 68. 
Kegley, S. et al (1999). Disrupting the Balance, Ecological Impacts of Pesticides in California, 

California, USA 
Hackenberg D (2007). "Letter from David Hackenberg to American growers. Plattform 

Imkerinnen Austria. 



142 Pesticides in the Modern World - Risks and Benefits 

Mills, L. S. Et al (1993) The Keystone-Species Concept in Ecology and Conservation, 

BioScience 43, (4), 219-224 
Miller, G. T. (2004), Sustaining the Earth, 6th edition. Thompson Learning, Inc. Pacific 

Grove, California, USA 
Rockets, R. (2007). Down On the Farm, Yields, Nutrients And Soil Quality 
Fox, J. E, et al (2007). "Pesticides reduce symbiotic efficiency of nitrogen-fixing rhizobia and 

host plants". Proceedings of the National Academy of Sciences, USA 
Johnston, A. E. (1986). "Soil organic-matter, effects on soils and crops". Soil Use Management 

2,97-105 
Lotter DW, Seidel R, and Liebhardt W (2003). "The performance of organic and conventional 

cropping systems in an extreme climate year". American Journal of Alternative 

Agriculture 18: 146-154 
Gilliom, R.J. et al (2007). The Quality of our nation's waters: Pesticides in the nation's 

streams and ground water, 1992-2001. US Geological Survey 
Bingham, S (2007), Pesticides in rivers and groundwater. Environment Agency, UK. 
Heather, B. et al (1997). Movement of Pesticides and Best Management Practices, Ground 

Water Pollution Primer, Virginia, USA 
Henry, L. (2003) Levels of some Pesticides in Environmental Samples from Southern Lake 

Victoria and its Catchments and their Chemodynamics in Tilapia Species, Water 

and Sediments Under Experimental Conditions, Tanzania 
Hayes et al, (2010) The cause of global amphibians decline: a developmental endocrinologist 

perspective, Journal of experimental biology, 213 (6) 921 
Muckenfuss A. E. et al, (1990). Natural mortality of diamondback moth in coastal South 

Carolina Clemson University, Coastal Research and Education Center 
Daly, H. et al (1998). Introduction to insect biology and diversity, 2 nd edition, Oxford 

University Press, New York, USA 
Sacramento, C. A. (2008). Department of Pesticide Regulation "What are the Potential 

Health Effects of Pesticides?" Community Guide to Recognizing and Reporting 

Pesticide Problems, 27-29 
Lorenz, E. S. (2009) "Potential Health Effects of Pesticides." Ag Communications and 

Marketing. 1-8 



8 



Ecological Impacts of Pesticides in 
Agricultural Ecosystem 

Khalil Talebi 1 , Vahid Hosseininaveh 1 and Mohammad Ghadamyari 2 

department of Plant Protection, College of Agriculture and Natural Science, 

University of Tehran, 
department of Plant Protection, College of Agriculture, University ofGuilan, 

Iran 



1. Introduction 

Pesticides are essential tools in integrated pest management (IPM) programs which can have 
the great influence if they are used properly. However, the adverse impacts of these 
compounds on the environment and ecosystem should not be ignored. The ecological effects 
of pesticides can be discussed from different points of view. Some of the significant 
consequences of use of pesticides are side effects of the pesticides on non-target organisms, 
sub-lethal effects of the pesticides on target and non-target organisms, emergence of resistant 
populations and pesticide residue and their entry into the trophic network. Side effect of the 
pesticides is a controversial issue in pesticides applications. They kill natural enemies present 
in the field and ecosystem and destroy the natural equilibrium between the hosts and their 
natural enemies. In the absence of natural enemies, pest populations increase rapidly and 
makes more controlling efforts, usually pesticides, necessary. In spite of pests, pesticide 
resistance in natural enemies is not common due to lower exposure to pesticides. Sub-lethal 
deposits of pesticides can change some biological traits of the organisms exposed to low and 
highly low concentrations of the toxicants. Sublethal impacts of pesticides are mostly ignored 
in ecological pesticide assessment because most pesticide assessments are performed as 
individual-level bioassays and population-level of toxicants has not been considered. Insects 
(pests and natural enemies) exposed to sub-lethal concentrations of pesticides show some 
changes in their life history's traits. Resistant populations emerge due to the misuse of 
pesticides. The populations with high ecological potential are gradually selected generation by 
generation and subsequent populations are remarkably or completely insensitive to pesticides. 
Resistant populations are usually different from natural population in their fertility life table 
characteristics. Nowadays, the existence of pesticides residue in agricultural crops and their 
entrance into the trophic network has endangered human health and environment, and it has 
also necessitated the correct use of the pesticides. In the current chapter, the most significant 
ecological impacts of pesticides in agricultural ecosystems have been discussed. 

2. Impacts of pesticides on natural enemies 

The concept of Integrated Pest Management (IPM) was initially defined as the combined 
use of natural enemies and pesticides to manage pests (Stern et al., 1959). The IPM 



144 Pesticides in the Modern World - Risks and Benefits 

concept later includes coordinated use of all possible tactics to suppress pest damage 
(Smith et al., 1976, as cited in Ruberson et al., 1998). Use of selective pesticides or rates, 
temporal separation of pesticides and natural enemies, and spatial separation of 
pesticides and natural enemies are three main area of integrating natural enemies with 
pesticides in pest management programs (Ruberson et al., 1998). Conventional use of 
insecticides can have deleterious effects on natural enemy populations because beneficial 
arthropods can have greater susceptibility to low concentrations of insecticides than their 
prey or host (Ruberson et al., 1998; Torres & Ruberson, 2004). Pesticide compatibility with 
biological control agents is a major concern to practitioners of IPM, and knowledge about 
the activity of insecticides toward pests, non-target insects and the environment is a 
necessity (Stark et al., 2004). 

Pesticides exert a wide range of lethal (acute and chronic) and sublethal (often chronic) 
impacts on natural enemies (Rezaei et al., 2007; Ruberson et al., 1998; Stark et al., 2004). 
Talebi et al. (2008) have published a comprehensive reviewed on the impacts of pesticides 
on arthropod biological control agents. Sublethal effects are expressed as some changes in 
the insect's life history attributes (Ruberson et al., 1998). Many studies have been performed 
on the evaluation of the toxicity of various pesticides to beneficial organisms (Kavousi & 
Talebi, 2003; Lucas et al., 2004; Medina et al, 2003; Oomen et al., 1991; Paine et al., 2011; 
Rezaei et al., 2007; Steiner et al, 2011; Urbaneja et al, 2008; Van de Veire et al., 2002; Van den 
Bosch et al., 1956; Walker et al., 1998). Some important issues including natural enemy 
species, life stages/ sexes, routes of pesticide entry, life history parameters, plot size for field 
screenings and pesticide formulations and rates must be considered for designing bioassays 
evaluating the effects of pesticides on natural enemies (Ruberson et al., 1998). One of the 
commonly used methods in testing the side effects of pesticides on natural enemies, 
recommended by the International Organization of Biological Control (IOBC), is a tiered 
approach whereby initial pesticide screening is done in the laboratory, and, depending on 
the results obtained, semi-field or field tests may be conducted (Dohmen, 1998; Hassan, 
1998). This method has been designed to evaluate the acute residual toxicity as well as 
sublethal effects of the pesticides on the reproductive performance (Vogt et al., 1992). In this 
method, dead subjects are recorded (often daily) and the total mortality is calculated. The 
value of mortality (M) for the treated series is determined as the corrected mortality 
according to Abbott (1925). The average number of progenies (R) is measured as fecundity 
affected by exposing to a pesticide. The total effect of a pesticide (E) is calculated by the 
formula E = 100% - (100% - M) xR proposed by Overmeer & Van Zon (1982). Based on the 
total effects, a pesticide is classified using IOBC evaluation categories (Sterk et al., 1999). 
Rezaei el al. (2007) investigated the effects of imidacloprid, propargite and pymetrozine in 
laboratory experiments using IOBC-system on the common green lacewing, Chrysoperla 
carnea (Stephens). All three tested pesticides produced significant adverse effects on pre- 
immaginal survival (p<0.01). Imidacloprid had no significant effect on fecundity, but 
propargite and pymetrozin caused significant reductions (p<0.05). According to IOBC 
classification, imidacloprid was found to be harmless (E=27.44%), propargite (E=49.78%) 
and pymetrozine (E=66.9%) were determined as slightly harmful. 

3. Population-level impacts of pesticides 

Sublethal effects of pesticides on the fitness of individuals are usually assessed using 
laboratory bioassays with insects (Grant, 1998) due to reduced variation among subject 



Ecological Impacts of Pesticides in Agricultural Ecosystem 145 

insects and high validity of statistical analyses (Robertson & Preisler, 1992). On the contrary, 
insecticide bioassays with field-collected insect subjects reduce reliability on the real effects 
of insecticides because of heterogeneity among tested individuals (Robertson & Worner, 
1990). In fact, the main research aim in ecotoxicological studies is predicting of insect field 
populations faced with sublethal concentrations/ doses of pesticides (Ferson et al., 1996). 
However, most studies deal with the impact of insecticides on individuals or some 
components of individuals (Stark & Banks, 2003). In laboratory tests, individual responses to 
chronic toxicity may be evaluated from morphological, biochemical, physiological, 
molecular and ecological point of view. Some responses such as reduction of growth or 
fecundity or increase in mortality rates or development times (Grant, 1998) are more easily 
measured in most insecticide bioassays. Although, mortality is the endpoint of interest for 
many acute studies and it may be used as an endpoint criterion in chronic exposure 
bioassays, reproductive inhibition or growth retardation are generally considered more 
sensitive measurements, particularly for the estimation of sublethal responses (Villarroel, 
1999; Stark & Banks 2003). Sublethal doses/ concentrations of toxicants may change life 
span, development rates, fecundity, egg viability, sex ratio, consumption rate and behavior 
of subjects (Dempster 1968; Ruberson et al., 1998; Stark & Banks, 2003, Stark & Rangus 1994; 
Stark et al. 1992a, 1992b; Vinson, 1974). Individual (sub-population) level responses and 
population level consequences can be related using life table response experiments (Caswell, 
1989). Data generated within life table response experiments give valuable information to 
the assessment of population-level consequences of toxicant sublethal effects (Caswell, 
1989b; 1996a, 1996b; Ferson et al., 1996; Grant, 1998; Stark & Banks 2003). 

3.1 Life table response experiments 

Lethal dose/ concentration of an insecticide that kills 50% of a population (LD50 or LC50) is 
commonly used as a simplistic criterion for determining and comparing the effects of 
toxicants (Stark et al., 2007). This approach relies on the death of individuals and ignores 
many consequent impacts of a toxicant on survivors. In addition to death, exposure to a 
toxicant may result in simultaneous manifestation of multiple sublethal effects (Stark & 
Banks, 2003; Stark et al., 2004). Under the phenomenon population compensation, if 
sublethal effects do not occur but the population density is reduced, survivors may have 
more resources available and actually produce more offspring than untreated populations 
(Stark et al., 2007). Effective concentration/ dose of a toxicant that affect x % of a population 
(EC X or ED X ) is also used when sublethal effects are scored. (Kammenga & Laskowski, 2000). 
Demographic toxicological analyses or life table response experiments is another approach, 
which takes into account total effects that a toxicant might have at the levels of organization 
higher than the individual (Stark et al., 2004). The advantage of this approach is that a total 
measure of the effect is determined that incorporates lethal and sublethal effects into a single 
endpoint, the intrinsic rate of natural increase or r m (Kammenga & Laskowski, 2000; Stark & 
Banks, 2003), which can detect subtle, individual-level effects of contaminants that alter the 
growth of populations at rates below the lethal concentration limits (Bechmann, 1994, as 
cited in Rezaei et al., 2007). The first life table response experiment was performed by Birch 
(1953) to study the impacts of temperature, moisture and food sources on flour beetles (as 
cited in Kammenga & Laskowski, 2000). The approach has been widely used in 
ecotoxicological studies; however few studies have been published on the use of 
demography and similar measures of the population growth rate for evaluating the effect of 
pesticides on insects, especially insect natural enemies (Kammenga & Laskowski, 2000; 



146 Pesticides in the Modern World - Risks and Benefits 

Rezaei et al., 2007; Stark & Banks, 2003). Life table response experiments are being increased 
to measure multiple endpoints of effects and have been recommended as a superior 
laboratory toxicological endpoint (Stark et al., 1997). In general, the main reason to use life 
table response experiments in toxicological studies is revealing of total effect (lethal, 
sublethal and too subtle impacts) of a toxicant on an insect at the population level. In a few 
investigations, especially in pesticide side effect studies, total effect of a pesticide is 
measured using the index E which incorporates mortality and fecundity (Overmeer & Van 
Zon, 1982; Rezaei et al., 2007). However, the index E is not like the demographic parameters 
(such as r,„) which measure the impact of a toxicant at population level. 

3.2 Construction of a life table 

Demography has been used in a small number of toxicological studies to evaluate lethal and 
sublethal effects of toxicants on insect populations (Stark & Banks, 2003; Stark et al., 2007). 
The basic principal in insect toxicological demography is construction a fertility table. The 
construction of a number of life tables is an important component in the understanding of 
the population dynamics of a species (Carey, 1993). 

A life table, for each treatment (toxicant concentration or dose), is constructed by following 
an insect cohort (egg, larva or adult), till the death of all individual members of a cohort, 
individually, and recording the age of each female (x), the probability that a new individual 
is alive at age x (L x ), and the number of female offspring produced by a female with 
attributed x (m x ) were recorded. Each individual from the initial cohort is treated according 
to a convenient procedure depends on test subject, toxicant and purpose. The survived 
individuals from the treated individuals are maintained and monitored individually to 
collect necessary data for construction life tables. 

The precise value of the intrinsic rate of increase (r m ) is obtained by solving the Euler 
equation (Andrewartha & Birch, 1954): 

f L x m x e- rx =1 (1) 

x=0 

In this equation, y is the oldest age class, L x is the survival of a newborn female to the 
midpoint of an age interval, and x is the age of each female at each age interval. In addition 
to r„v the other main fertility life table parameters including net reproductive rate (R ), 
generation time (T), doubling time (DT), and finite rate of increase (A) are also computed 
using the following formulas, respectively: 

R =£L x m x (2) 

T = £ xL x m x/Z L x m x (3) 

DT = ln(2)/r m (4) 

X = e r ™ (5) 

In fact, these parameters are estimations for a given population; therefore, the uncertainty 
associated with them must be estimated. Uncertainty associated with the parameters can be 
estimated using two techniques; jackknife and bootstrap. However, jackknife technique is 



Ecological Impacts of Pesticides in Agricultural Ecosystem 147 

more popular and nearly all estimations are performed according to this method. The 
jackknife technique is used for ease of statistical comparisons among life table parameters 
related to each treatment and for estimating the standard errors (SE) associated with the 
parameters. First, the precise value of r m is calculated for all of the raw data (r to tai)- Then, one 
of the insect subjects is omitted and an r m is computed for the remaining insects (n-1). Based 
on the suggested equation by Meyer et al. (1986) the jackknife pseudo-values were 
calculated for this subset of the original data according to: 



'total 



-(n-l)r t (6) 



The value of n is the number of insects needed to construct a fertility life table. This process 
is repeated until pseudo-values were calculated for all n possible omissions of one insect 
from the original data set. Finally n number of calculated f ; are provided to calculate the 
mean (r,) and its SE. 



1 " 

r r -Zn (7) 



SE(rj) = MM (8) 



In the equation 8, sf is the variance of the n jackknife pseudo-values. This algorithm is used 
for estimating uncertainties associated with the four other parameters. All jackknife 
pseudovalues for each treatment are usually subjected to analysis of variance (ANOVA) 
followed by a convenient mean comparison test. The nonparametric tests are also used for 
some pseudovalues which are not meet ANOVA perquisites (Rezaei et al., 2007). 

3.3 Life table parameters 

Intrinsic rate of natural increase, r,„, is the main and the best estimator for growth rate of 
insect populations. When values of r„, are positive, a population is increasing exponentially; 
when r,„ is equal to zero, a population is stable and when r,„ is negative, a population is 
declining exponentially and headed toward extinction (Kammenga & Laskowski, 2000). In 
toxicological studies, values for r m are statistically compared among different cohorts 
(toxicant-treated and control). Rezaei et al. (2007) in life table response experiments of C. 
carnea with some pesticides revealed that imidacloprid and propargite had no significant 
effects on the intrinsic rate of natural increase, while pymetrozine caused a 34% reduction in 
r„, value (p<0.05). Propargite was non-toxic to C. carnea under the tested conditions. The life 
table assay showed more adverse effects of pymetrozine than a non-life table response 
experiment method (IOBC method). Lashkari et al. (2007) studied the efficiency of 
imidacloprid and pymetrozine on population growth parameters of cabbage 
aphid, Brevicoryne brassicae L. (Homoptera: Aphididae). They revealed that r m were lower in 
imidacloprid and pymetrozine treatments than in controls. In such investigations, simple 
statistical comparisons of r,„ values among cohorts determine efficiency of toxicants. 
However, a more precise and complicated method is estimating of a concentration/ dose of a 
toxicant at which r,„ value is reduced by 50% (population-level EC50 or ED50) or specific 
proportions (population-level EC X or ED X ) under laboratory conditions. (Suter & Glenn, 
1993; Tanaka & Nakanishi, 2001) . 



148 Pesticides in the Modern World - Risks and Benefits 

3.4 Age-stage two-sex life table 

In construction of a fertility life table, raw data is commonly collected from survival and 
reproduction of female individuals. In this method, males are completely ignored and only 
used for fertilizing females in a cohort. Ignoring the sex of individuals can result in errors 
(Chi, 1988). Chi & Liu (1985) and Chi (1988) developed a new method, age-stage two-sex life 
table, for construction a life table with taking into consideration both female and male sexes. 
In a small number of investigations, two-sex life table theory have been used for 
construction of the life table and data analysis (Chi & Su, 2006; Kavousi et al., 2009; Refaat et 
al, 2005; Schneider et al., 2009; Yang et al, 2006; Yu et al., 2005). As far as the authors aware, 
there is only one investigation, Schneider et al. (2009), on the effect of a toxicant on an insect 
according to the age-stage two-sex life table theory. Schneider et al. (2009) determined the 
side-effects of glyphosate (a herbicide) on development, fertility and demographic 
parameters of C. externa (Neuroptera: Chrysopidae) in the laboratory. They revealed that 
glyphosate will decrease arthropod population performance and the major detrimental 
effect observed on C. externa was on fecundity and fertility. 

3.5 Drawbacks to the use of life table response experiments 

Although life table response experiments may provide the most complete data for the 
impacts of a pesticide on an animal subject at population-level, there are some 
disadvantages associated with this method. The most important one is that life table 
response experiments are expensive and time consuming. Construction of a life table is 
difficult or impossible for some species (long-lived species) when exposed to a pesticide 
because of the low rate of reproduction. The other major disadvantage is unrealistic 
conditions under which a life table is constructed. These conditions are far from the natural 
conditions in field (Kammenga & Laskowski, 2000; Stark & banks, 2003). 

4. Resistance of pests to pesticides 

Pesticides are used extensively for control of invertebrate pests, plant pathogens, weeds and 
rodents and other pests in a wide range of crops and for veterinary purpose. Resistant to 
pesticides develop in insects, mites, fungi, weeds, bacteria and rodents. Repeated 
applications and extensive use of the synthetic pesticides has toxicity toward natural 
enemies and cause resistance development in pest species against major classes of pesticides 
throughout the world. The repeated and extensive application of pesticides caused majority 
on susceptible individuals in population and only some resistant individuals survive from 
pesticide exposure. The offspring genotype of survival individual is homozygous or 
heterozygous that depends on history of pesticide application and type of pesticides. The 
offspring inherit the resistant genes and survival ability from the exposure to the pesticides. 
The surviving individuals multiply in absence of their natural enemies and finally replace 
the non-resistant population. The development of pesticide resistance is a Darwinian 
evolutionary process at a rate that rare genes conferring resistance to pesticides are selected 
by the high selection of pesticides. Resistance to pesticides is defined as "the development of 
an ability in a population of a pest to tolerate doses of pesticides that would prove lethal to 
the majority of individuals in a normal population of the same species" (Stenersen, 2004). 
The first case of resistance occurrence in insect pests was reported in 1908. This document 
reported the failure in the control of Quadraspidiotus perniciosus (Hem.: Diaspididae) by 
sulphur. After this report, Melander (1914) reported resistance of three scale strains in 



Ecological Impacts of Pesticides in Agricultural Ecosystem 149 

United State to sulphur and sulphur-lime (as inorganic pesticide) (as cited in Stenersen, 
2004). The organochlorine and synthetic insecticides were commercialized for chemical 
control of pests in the 1940's. The first case of DDT resistance in insect was reported in 
Musca domestica few years after introduction. After that, new insecticides such as 
cyclodienes, pyrethroids, organophosphates (OP), carbamates, formamidines, Bacillus 
thuringiensis, avermectins, spinosyns, insect growth regulators (IGR) and neonicotinoids 
were introduced for pest control and the cases of resistance to these compounds appeared a 
few years after their application. Now, more than 504 key pest species were resistant to 
pesticides and the resistance to pesticides has become a major contemporary problem in pest 
management programs (IRM) worldwide. Stuart (2003) reported resistance of 520 insect and 
acari species, 150 plant pathogen species and 273 weed species to pesticides. 
Pesticides resistance reduces the ability control of pesticides on pests and leads to higher 
application rates to achieving satisfactory pest control. Pimentel (2003) estimated the major 
economic and environmental losses due to the application of pesticides on crops and 
veterinary purpose in the USA and showed the following costs: "public health, $1.1 billion 
year-1; pesticide resistance in pests, $1.5 billion; crop losses caused by pesticides, $1.1 
billion; bird losses due to pesticides, $2.2 billion; and ground water contamination, $2.0 
billion" (Pimentel, 2005). 

4.1 Detection and monitoring of resistance 

Reduced pesticide selection pressure for each resistance mechanism is necessary for 
avoiding and delaying control failure prior to occurrence of resistance. For achieving this 
purpose, successful detection techniques are required for avoiding resistance developing 
and a control failure. "These techniques could be able to detect of resistant individual at low 
frequency in natural population" (Scott, 1995). Techniques for monitoring resistance to 
different pesticides in pest population gathering valuable information for insecticide 
resistance management (IRM) employer. 

Detection and identification of resistance mechanisms to pesticides require monitoring 
approach with appropriate bioassay method. Monitoring of resistance is required in order to 
sustainable management of pesticide resistance and to know the status of resistance. 
Therefore, developing precision and reliable susceptibility test must be developed. These 
tests must be also accurate, chip, easy to perform in variety of conditions in laboratory and 
on farm site. So far, many standardized susceptibility test method were presented by Food 
Agriculture Organization (FAO) and World Health Organization (WHO) such as exposure 
to standard residue treatment on glass scintillation vial or filter paper, plastic bags, topical 
application, spray application of standard solutions, and resistance detection kits and strip. 
Resistance frequencies can be detected and monitored by bioassays using diagnostic 
(discriminating) dose (LD99) and estimating resistance factor (Rf= LD50 of resistant 
population/ LD50 of susceptible population). The diagnostic dose (ie. LD99) can be calculated 
from regression line of log dose probit-mortality data using appropriate software such as 
POLO-PC. This dose discriminate the tested population as susceptible and resistant and the 
pests that die after exposures with LD99 of pesticide are classified as susceptible and those 
individual that survive from exposure considered as resistant. The discriminating-dose 
assay is a chip, less time consuming approach for monitoring resistance in natural pest 
populations. These bioassay procedures provide valuable data for monitoring of resistance 
but this method is not practical for detection of resistance in low frequencies in field 
population of pests (Roush and Miller, 1986). 



150 Pesticides in the Modern World - Risks and Benefits 

The mechanisms of resistance are behavioral, reduced penetration, metabolism of toxicant to 
inactive product and target site insensitivity. These mechanisms can be detected using 
biochemical assay techniques (spectrophotometric and fluorometric methods) and 
molecular assays (base on DNA diagnostic) in one individual or small number of insect. 
Identification of resistance mechanisms is critical for determining of the cross resistance 
spectrum (Brogdon and McAllister, 1998). 

"Molecular methods and traditional assays (ie. bioassay) used for distinguish heterozygotes 
(SR), homozygous susceptible (SS) and homozygous resistant (RR) genotypes" (Scott, 1995). 
The environmental conditions such as temperatures, humidity, pH and light increase errors 
in biochemical and bioassay results but these conditions can not affect the results of 
molecular methods (Scott, 1995). Now, PCR-based techniques have been designed for field 
detection of modified acetylcholinesterase (AChE) and knock down (Kdr) in individual Myzus 
persicae (Field et al., 1996). "The amplified E4 or FE4 genes can be identified by restriction 
enzyme analysis or polymerase chain reaction (PCR)-based methods" (Field et al., 1996). 

4.2 Mechanisms of resistance to pesticides 

Biochemical and molecular basis of resistance mechanisms to pesticides in insects, acari, 
fungi, bacteria, weeds and vertebrate pests are similar. An exhaustive knowledge on 
biochemical and molecular resistance mechanisms in pests are useful for designing 
insecticide resistance management (IRM) strategies. Also, identification of resistance 
mechanisms is necessary for developing discriminating techniques for detecting and 
monitoring resistance genes and cross resistance spectrum in the field populations of pests 
(Hammock and Soderlund, 1986). The factors affecting pesticides effectiveness were 
distinguished in two classes: The first class decreases the amount of pesticide dose in action 
site including behavioural resistance, reduced penetration or adsorption, sequestration and 
detoxification. The second class is decreased target site sensitivity to pesticides that reduce 
the affinity of target protein toward activated pesticide (van leeuwen, et al., 2009). "In 
practice, probably more than 90% of all resistance cases in insects and mites are caused by a 
less sensitive target site and/ or an enhanced pesticide detoxification" (Roush and 
Tabashnik, 1990 as cited in van leeuwen, et al., 2009). The relative importance of these 
mechanisms depends on pest species and history of chemical application. 

4.2.1 Genetic mechanisms 

Genetic mechanisms of pesticide resistance involve some point mutations in genes and their 
over expression. These mechanisms were elucidated as follow: 

4.2.1.1 Gene amplification 

Devonshire and Moores, 1982 showed that the gene amplification of one of two closely 
related carboxylesterases (E4 and FE4) in M. persicae were associated with resistance to OP, 
carbamates and pyrethroids. Carboxylesterases sequester or degrade carbamate and OP 
insecticides before they reach to AChE in the nervous system. E4 and EF4 overproduction in 
resistant strains of M. persicae is due to amplification of structural genes encoding these 
enzymes (Field et al., 1988). 

4.2.1.2 Up- and down-regulation 

The research showed that cytochrome P450 enzyme were over expressed in some resistance 
strain of M. domestica through the increase of gene transcription by up-regulated 



Ecological Impacts of Pesticides in Agricultural Ecosystem 151 

mechanism. The up-regulation of a cytochrome P450 enzyme led to resistance when an 
insecticide is used in its toxic form on M. domestica. If pro-insecticide, i.e. a chemical must be 
converted in pest through metabolism to the active form, used against M. doimestica, down- 
regulation of cytochrome P450 or other metabolizing enzymes will increase resistance (Scott, 
1995). 

4.2.1.3 Structural change in insecticide- target molecules 

AChE, the gamma-aminobutyric acid (GABA) receptor, Voltage-gated sodium channels, 
nicotinic acetylcholine receptor, octopamine receptor and the juvenile hormone (JH) 
receptor are known as targets of pesticides and substitution of amino acid residues in these 
sites led to insensitivity of structural protein toward pesticides (Kono and Tomita, 2006). 

4.2.2 Behavioral resistance 

"Behavioral mechanisms, defined as evolved behaviors that reduce an insect's exposure to 
toxic compounds or that allow an insect to survive in what would otherwise be a toxic and 
fatal environment" (Sparks et al.,1989). There is a little literature on behavioural resistance 
mechanisms in insect due to difficulties in detection (as cited in Jensen, 2000). It seems the 
significance of this mechanism for resistance is less than other resistance mechanisms. 

4.2.3 Reduced penetration 

Reduced penetration of insecticide as a resistance mechanism has been studied 
in few insect species such as Leptinotarsa decemlineata. Reduced insecticide penetration via 
cuticle led to decrease the amount of dose in action site. The resistance ratio by this 
mechanism was lower than 3-fold (Scott, 1990), but because several different mechanisms 
are responsible for resistance to an insecticide and multiple resistance mechanisms may 
co-exist in an insect and act either additively or synergistically. 

Patil & Guthrie, 1979 compared the composition of the cuticular lipids of two resistant 
strains of M. domestica and their results showed that "total lipids, monoglycerides, 
diglycerides and sterol esters, sterols, fatty acids and phospholipid phosphorus were higher 
in resistant strains than in the susceptible strain". 

Three methods for detecting of this mechanism include: Wash-off, diffusion cell and disk 
technique. In wash-off radiolabeled insecticide was topically applied to the insects and 
then, at fixed times after application, un-penetrated insecticide was washed off with an 
appropriate solvent and quantified (as cited in Jensen, 2000). 

4.2.4 Metabolism of toxicants 

Three enzyme groups involved in metabolic resistance to pesticides: esterases, glutathione 
S- transferases (GST) and mixed function oxidaes (MFO). The following technique can be 
used for detection of these mechanisms. 

4.2.4.1 Esterase 

Esterases metabolize a variety of pesticides such as OP, carbamate, pyrethroids with ester 
linkages. "These enzymes confer resistance to pesticides in over 50 species of insects, ticks 
and mitesa" (Devorshak and Roe 1998; as cited in van Leeuwen et al., 2009). Detection 
and investigation of esterases-based mechanism can be achieved from synergistic bioassays 
and biochemical assays. For synergistic bioassays, some synergists such as DEF 
(S,S,Stributylphosphorotrithioate), TPP (0,0,0-triphenylphosphate), and IBP (0,0-bis[l- 



152 Pesticides in the Modern World - Risks and Benefits 

methylethyl] S-phenylmethylphosphorothioate) were used to inhibit esterases (Raffa & 
Priester, 1985 as cited in Jensen, 2000). However, the synergistic bioassays are useful to 
achieve valuable data, but DEF in higher concentrations inhibits MFO and esterase activity 
(Scott, 1990). 

In biochemical assay, increased esterase activity in resistant insect can be checked with some 
artificial substrates such as a- and fi- naphthyl acetates, a- and fi- naphtyl butyrates, a- and 
fi- naphtyl propionates and p- nitrophenyl acetates. These substrates hydrolyzed by general 
esterases and the involvement of esterase in resistance must be checked by more than one 
substrate. Also, the specific elevated esterase can be detected with immunological methods 
and an antiserum for example antiserum of E4 carboxylesterase M. persicae (Devonshire et 
al, 1986). An affinity purified immunoglobulin G (IgG) fraction from this antiserum has 
been used in a immunoplate assay to distinguish between the different resistant strains of 
M. persicae (Devonshire et al., 1986). 

4.2.4.2 GST 

The GSTs are involved in the detoxification of a wide range of xenobiotics including 
insecticides, fungicides, acaricides and herbicides (Salinas & Wong, 1999). GSTs catalyze the 
conjugation of hydrophobic electrophile compounds such as pesticides and their metabolites 
with the thiol group of reduced glutathione (GSH) (Habig et al., 1974). 

Elevated GST activity has been associated with resistance to the major classes of pesticides 
and the involvement of GSTs in resistance to insecticides is well reviewed in Enayati et al., 
(2005). Because GSTs can metabolize a wide variety of xenobiotics such as insecticides and 
plant allelochemicals, increased GST activity may be due to exposure of insect with foreign 
compound in environment not resistance mechanisms. Diethyl maleate (DEM) is used as 
synergist for inhibiting of GSTs involved in resistance in bioassays. 

GST activity can be measured from direct measurement of the conjugation of reduced 
glutathione with non-fluorescent monochlorobimane (MCB), l-chloro-2,4-dinitrobenzene 
(CDNB) and 3,4-dichloronitrobenzene (DCNB) as substrates using a spectrophotometer at 
340 ran. 

4.2.4.3 Cytochrome P450 

Cytochrome P450 plays important role in metabolism of pesticides and confers resistance to 
many classes of pesticides in mite, insect, weeds and fungi. These enzymes widely 
distributed in fungi, bacteria, yeast, insect, mite, weeds and invertebrate. Piperonyl butoxide 
(PBO) and sesamex was used in synergistic bioassays for involvement of MFO in resistance. 
However, PBO can inhibit both MFO and esterases-based resistance mechanisms in some 
insect and mite species (Gunning et al., 1999; Young et al., 2005). 

There are many P 45 o-monooxygenase isoenzymes with different substrate specificity within 
an insect. Therefore, in measuring activity of MFO- based resistance mechanisms must use 
different substrates and methods (Rose et al., 1991). Different biochemical methods have 
been used to study P 45 o-monooxygenase activity in insects and mites. One of these methods 
is measuring the total amount of heme containing protein using a heme-peroxidase assay 
(Brogdon et al., 1997). Another method uses the aldrin as substrate to measure P450- 
monooxygenase activity. O-demethylase can be detected using p-nitroanisole as substrate 
using spectrophotometer. O-deethylation activity of the artificial substrates, 7- 
ethoxycoumarin (7-EC) and ethoxy-4-trifluoromethylcoumarin, by MFO can be measured 
with fluorometric microplate assay (van Pottelberge et al., 2009). 



Ecological Impacts of Pesticides in Agricultural Ecosystem 153 

4.3 Fitness costs associated with pesticide resistance 

Genetic changes confer insecticide resistance in insects can affect their developmental time 
and reproductive potential. Resistance genes can alter some life table and physiology 
parameters of pests and thus causing a fitness cost (as cited in Gazavi et al., 2001). In some 
insect and mite species, genetic changes that enhance survival due to pesticides exposure 
reduce pest fitness in the absence of pesticides (Roff and Derose, 2001; Higginson et al., 
2005). Because insecticides caused a huge selection on pest population in a short time, 
therefore susceptible and resistant populations are useful models for evaluating fitness 
trade-off (Crow, 1957; McKenzie, 1996; Higginson et al., 2005; Ghadamyari et al., 2008). 
Mutation associated with insecticide resistance can disturb pest physiology (MacCarroll et 
al, 2000). 

Resistant and susceptible strains differ in some properties due to their adaptation to 
insecticides, such as developmental time, fecundity and fertility, overwintering success and 
sensitivity to alarm pheromone. Differences in the biological parameters affecting the net 
reproductive rate of insect populations are important for insecticide resistance management 
(IRM) (Haubruge and Arnaud 2001). Altered acetylcholinesterase (AChE) and esterase- 
associated resistance in peach aphids have life history disadvantages compared with 
susceptible counterpart (Ghadamyari et al., 2008). Also, altered GABA receptor, esterase, 
MFO and GST associated abamectin resistance in Tetranychus urticae showed life history 
disadvantages. 

Field and laboratory studies on different strains of M. persicae have been showed that 
adverse selection by pesticides caused "poorer winter survival, maladaptive behaviour and 
reduced reproductive fitness" (Foster et al., 2000). 

4.3.1 Maladaptive behavior 

Some resistance mechanisms can confer more fitness disadvantages to resistant strains than 
others. Some strains of M. persicae with over expressing high levels of carboxylesterase (due 
to structural gene amplification) show a reduced tendency to move away from senescing 
leaves compared with susceptible counterpart (Foster et al. 1997, 2003). This behaviour 
caused higher mortality during worst winter weather conditions and so can be regarded as a 
deleterious pleiotropic effect of pesticide resistance. Studies have been shown that peach- 
potato aphids caring both gene amplification and the knock down mutation has reduced 
response to alarm pheromone (Foster et al., 2003). 

4.3.2 Reduced reproductive success 

Many experiments measured reproductive fitness in the absence of pesticides in resistant 
and susceptible strains. The results of these experiments showed that individuals carrying 
resistant genes have lower reproductive rate than susceptible. When pressure of insecticide 
is diminished on resistant strain, the number of resistant individual in population quickly 
reduce due to fitness cost (Crow, 1957; Carriere and Roff, 1995; McKenzie, 1996; as cited in 
Arnaud et al., 2005). Although the majority of insecticide resistance strains show fitness cost 
(McKenzie, 1996; Foster et al., 2003; Berticat et al., 2004; as cited in Arnaud et al., 2005; 
Ghadamyari et al., 2008), a few researches present species with no fitness cost (McKenzie, 
1996; Oppert et al., 2000; McCant et al., 2005). For example "some resistant strains of 
mosquitoes in absence of pesticides showed only one quarter of the reproductive potential 
of susceptible strains" (Georghiou and Taylor, 1977). Varroa destructor has little or no 
reproductive fitness cost associated with pyrethroid resistance in Texas (Martin et al, 2002). 



154 Pesticides in the Modern World - Risks and Benefits 

Tetranychus urticae resistance to abamectin showed reduced reproductive success compared 
with susceptible populations and the r m of susceptible population was higher than r m of 
resistant population (unpublished data). 

In contrast, in Tribolium castaneum "resistant to malathion, susceptible male individuals 
show reduced reproductive success compared with resistant lines" (Arnaud et al., 2005). 
Foster et al. (2000) showed reduced reproductive success in M. persicae expressing the 
highest levels of carboxylesterase. 

4.3.3 Reduced overwintering ability 

Winter field trials by Foster et al., 1996 showed that UK M. persicae clones expressing high 
levels of esterase-based resistance (i.e. R2 and R3) present higher mortality than their 
susceptible (S) and -Rl counterparts during worst weather conditions (Foster et al., 1996). In 
Heliothis virescens, resistance to CrylAc is recessive and associated to cadherin gene (Morin 
et al, 2003), research showed fitness costs associated resistance affecting overwintering 
success and survival on non-Bt cotton (Carriere and Tabashnik, 2001). The frequency of pink 
bollworm resistance to Bt cotton has not increased in the field compared with laboratory 
that show fitness cost (Tabashnik et al., 2003). Monitoring Culex pipiens mosquitoes 
overwintering in a cave in southern France (in an area where OP insecticides are widely 
used) showed a "decrease in the frequency of insecticide-resistant mosquitoes compared 
with susceptible counterpart, indicating a huge fitness cost" (Gazave et al., 2001). In the pink 
bollworm, Pectinophora gosypiella, has been shown fitness costs at low temperatures 
associated with resistance to Bt and this can delay resistance of this pest to Bt cotton 
(Carriere et al., 2001; Tabashnik et al., 2005). 

4.3.4 Why insecticide resistance caused a fitness cost? 

The occurrence of fitness costs in insecticide resistant strain is reported for many pests such 
as M. persicae (Ghadamyari et al., 2008), T. urticae (unpublished data) and Sitophilus zeamais 
(Coleoptera: Curculionidae) (Arau'jo et al., 2008a, 2008b; Guedes et al., 2006). Populations of 
T. urticae, S. zeamais and M. persicae with different levels of resistance to different pesticides 
have shown to be good subjects and models for evaluating the physiological base of fitness 
cost associated pesticide resistance. 

Recently, some attempt was done to show the relationship between the energy consumption 
and the energy reserves available for metabolism of pesticides. The energy consumption 
could be measured using the electron transport activity (at the mitochondrial level), while 
reserve energy for metabolism could be achieved by measuring total lipids, protein and 
sugar contents by spectrophotometric method. The differences between energy 
consumption and the energy reserves represent the energy available for growth and 
biomarker of fitness cost in resistant populations. Our research on fitness cost of T. urticae 
resistant to abamectin showed that no significant differences were presented between the 
amounts of fuel nutrients macromolecules (carbohydrate, protein and lipid) in the resistant 
and susceptible populations of T. urticae, the amount of energy consumed was higher for 
resistant population when compared to its susceptible counterpart. Also the susceptible 
population exhibits a significantly higher r,„ than the resistant population. These suggested 
that the resistant population may be less fit than the susceptible compartment. 
The following theory was presented by Guedes et al., 2006, Arau'jo et al., 2008a, 2008b and 
Lopes et al., 2010 and we attempt to discuss their theory with their justifying fitness cost in 
S. zeamais. 



Ecological Impacts of Pesticides in Agricultural Ecosystem 155 

Populations of S. zeamais with different levels of susceptibility to insecticides were used a 
model for evaluating the mechanisms of fitness cost associated resistance. Demographic and 
competition studies carried out on different strains of S. zeamais susceptible and resistant to 
pyrethroids showed fitness costs associated with insecticide resistance in some strains and 
no fitness costs in other strain (Fragoso et al., 2005; Oliveira et al., 2007). 
The susceptible, resistant no-cost and resistant cost strains showed some differences in some 
biochemical parameters as follow: 

I. Some differences in the accumulation and consumption of fuel nutrients 
macromolecules were observed between S. zeamais pyrethriod-resistant and susceptible 
strains. These differences caused S. zeamais could be able detoxify insecticides without 
reduction in its reproductive potential. Pyrethroid- resistance cost strain has greater 
stored total proteins and carbohydrates compared with susceptible and resistant cost 
strains (Arau'jo et al, 2008a, 2008b). Finally Arau'jo et al. (2008a, 2008b) concluded that 
increased energy reserves may be due to increased digestive enzyme activities. 

II. The pyrethroid -resistant strains showed increased serine- and cysteine-proteolytic and 
cellulolytic activity. Also, kinetic parameters of these enzymes were different in 
susceptible, resistant no-cost and resistant cost strains. These differences suggested that 
cysteine-proteinase and cellulase activities were more important for justifying the cost 
of insecticide resistance in S. zeamais strains (Arau'jo et al., 2008b). The activity of 
carbohydrases specially amylase was higher in the resistant no-cost strains suggesting 
that a more efficient energy storage may justify the fitness costs due to the over 
expression of detoxify enzymes (Lopes et al., 2010). 

III. The pyrethroid-resistant no-cost strain of S. zeamais show higher grain loss, higher 
respiration rate, higher body mass, and larger energy reserve cells than the pyrethroid- 
resistant cost strain and susceptible strains (Guedes et al., 2006). These advantages 
cause resistant no-cost strain has additional reserved energy for detoxifying insecticides 
without any adverse effect on their life table parameters (Guedes et al., 2006). 

Some resistant strains of green peach potato aphid in the UK showed various fitness costs, 
such as reduced overwintering ability, lower rate of movement away from senescing plant 
leaves at low temperatures, reduced responses to alarm pheromones and reduced 
reproduction (Foster et al, 2000, 2002, 2005; Ghadamyari et al, 2008). Foster et al., 2000 
concluded that differences in behavior reducing M. persicae survival due to pleiotropic 
effects of the kdr mechanism and over expression of E4 and FE4 gene (Foster et al., 2000) 
Also behavioural characteristics is associated with knock down resistance in the M. domestica 
(Foster etal., 2003). 

5. Pesticides residue in the environment 

Chemical pesticides are used to control target pests. Extensive use of pesticides after World 
War II has substantially increased the agricultural production. However non target 
organisms including human and wildlife are affected. Pesticides are bioactive molecules that 
interfere with vital biochemical and physiological processes in organisms. Some are lethal to 
exposed organisms and many can cause disorder at sub lethal level. Extensive research is 
necessary to clarify the side effects of pesticides on organisms. About 3 billion kg of 
pesticides is applied each year with a purchase price rose to $47 billion in 2008, worldwide 
(Pimentel, 2009; Frabotta, 2009). 



156 Pesticides in the Modern World - Risks and Benefits 

The environmental persistence is different from pesticide to pesticide. Some are persistent 
and remain in the environment either as a parent compound or transferred products. The 
fate of pesticides in soil depends on the value of Koc, carbon sorption coefficient. High 
values of Koc indicate a pesticide that strongly adsorbs to the soil particles and less likely to 
move with water. Moreover, soil composition, pH, moisture content and microbial activity 
affect pesticide persistence. 

5.1 Insecticides 

The most toxic and environmentally persistent compounds are found among insecticides; 
therefore, the emphasis is on groups of insecticides that have been studied in detail. 

5.1.1 Chlorinated insecticides 

The first synthetic insecticide was DDT with a wide spectrum of insecticidal action that was 
used in agriculture and against insect vectors of deadly diseases. DDT solubility in water is 
very low, about 0.006 mg/1, which makes it one of the most hydrophobic insecticides. DDT 
residues either as parent compound or its metabolites DDD and DDE are stable and have 
high persistence in the environment. It has a great tendency to be stored in fatty tissue of 
different organisms. After the introduction of DDT, HCH was marketed. HCH has eight 
isomeric forms of which y-isomer is called Lindane. Lindane is a volatile insecticide and was 
used against agricultural and households pests. Lindane is less persistent than the other 
organochlorine insecticides especially under moist conditions. The cyclodiens are stable 
organochlorine soil applied insecticides. These included aldrin, dieldrin, endrin, chlordane, 
heptachlor and endosulfan. Cyclodiens are environmentally persistent compounds that 
have raised concern about adverse effects on human health and wildlife. Residues of DDT 
and its metabolites DDD and DDE, dieldrin and heptachlor epoxide were detected in high 
percentage of soil and water samples from agricultural areas decades after their use were 
banned. Extensive studies on organochlorine pesticides has shown the environmental 
persistence of these Compounds (LeaMond et al. 1992; Reiser & O'Brien, 1999). 

5.1.2 Organophosphorus Insecticides 

Organophosphorus insecticides replaced persistent organochlorine compounds. Utilization 
of these insecticides increased rapidly and for several decades comprised high proportion of 
total insecticide use. Organophosphates are unstable compounds, however some of these 
insecticides are more acutely toxic to invertebrate than chlorinated insecticides. Parathion 
was the first marketed product that was effective against a wide variety of pests. Some 
organophosphates caused severe toxicity associated with many deaths especially in 
developing country, whereas a few compounds such as malathion are relatively safe to 
mammals and degrade fairly rapidly in the environment. Most organophosphates are 
harmful to beneficial arthropods, though few compounds such as phosalone and 
dimethoate are considered as harmless compounds. 

The occurrence and movement of some organophosphate pesticides are reported in rivers 
and streams. Several studies conducted to find out the presence of organophosphate 
residues in California rivers during 1993-1994. Diazinon, methidathion, dimethoate and 
chlorpyrifos residues were detected in water samples. The detection occurred mostly during 
rainy season, showing how run off influences the presence of pesticide residues in rivers 
and streams (Ganapathy et al. 1997). 



Ecological Impacts of Pesticides in Agricultural Ecosystem 157 

5.1.3 Carbamate Insecticides 

Carbamate insecticides are a group of synthetic compounds derived from carbamic acid. 
The first carbamate carbaryl was an N-methyl carbamate with high insecticidal activity 
against many insect pests and ectoparasites of animals. Carbamates especially N-mathyl 
carbamates are extremely toxic to hymenoptera and are lethal to exposed foraging bees. 
Carbamates biodegradation in environment is relatively rapid. 

Oxime carbamates are a group of carbamate with systemic action. Aldicarb, an oxime 
carbamate is the most potent toxic substance (LDso=0.9 mg/kg) ever used in crop protection. 
Because of high toxicity it is used as granular formulation. Aldicarb sulfoxide is its oxidative 
metabolite that may undergo further oxidation to the sulfone. Oxidative residues and its 
parent compound (Tatal aldicarb) are toxic and highly mobile in the environment. Total 
aldicarb is detected especially in shallow ground water since 1979. Ground water quality 
monitoring has shown that many samples contain aldicarb residues and some of them 
exceeded maximum acceptable concentration (Priddle et al. 1989; Marade & Weaver 1994). 

5.1.4 Pyrethroids 

Pyrethroids are synthesized based on the model of naturally occurring pyrethrins with more 
stability to light and air. Pyrethroids are used in agriculture, homes, restaurants and 
hospitals. These compounds are readily metabolized by man but they are effective against 
insects. Most pyrethroids are esters however non-ester pyrethroids are discovered with 
good insecticidal activity and low mammalian toxicity. These readily penetrate insects and 
paralyze their nervous system (Reigart et al v 1999). Since pyrethroids are highly toxic to 
insects, both the beneficial and pest insects are affected. 

Sunlight, microbial activity, heat, and moisture accelerate pyrethroids break down, hence in 
areas with limited sunlight, pyrethroids persist for a long time. After treatment in the home, 
cypermethrin persist for about three months (Wright et al, 1993). Pyrethroids are lipophilic 
compound that strongly absorb to colloids of soil. Dissipation of cypermethrin, fenvalerate, 
and deltamethrin, were investigated in yellow red soils. The half-life of theses compounds 
were 17, 19, 18 days in unsterilized, compared to 76, 92 and 80 days in sterilized soil (Gu et 
al, 2008). This experiment shows the effects of biodegradation in pyrethroids life span in 
soil. 

5.1.5 Neonicotinoids 

Neonicotinoids are similar to nicotine with the same mode of action. These insecticides have 
been used worldwide. Most neonicotinoids are absorbed and translocated to the tips of the 
plants. Imidacloprid is the first widely used insecticide of this group with relatively low 
mammalian toxicity. However, it is harmful to beneficial arthropods including bees 
(LD5o=0.008 u.g /bee). Imidacloprid and clothianidin are more toxic to bees as spray than as 
seed dressing (Tennekes, 2010). Most neonicotinoids are moderately soluble and so they are 
mobile in the environment. In ground water 18 feet below sandy loam soil concentrations of 
imidacloprid ranged from < 0.1 ppb to 1 Ppb (Bacey, J. 2000). This observation shows the 
potential of imidacloprid to leach downward into shallow groundwater. Imidacloprid has a 
moderate binding affinity to soil colloids. Half-life in soil varies under different conditions. 
The half -life of imidacloprid in soil was 48-90 days, depending on the ground cover (Scholz 
& Spiteller, 1992). Laboratory experiments showed that persistence of another 
neonicotinoid, thiamethoxam is highly depending on moisture and the half-life varied from 



158 Pesticides in the Modern World - Risks and Benefits 

45 to 300 days (Gupta et al. 2008). The half -life of neonicotinoids increases with increasing 
soil colloids. Overall, neonicotinoids have a low potential to persist in soil and accumulate 
in the environment. 

5.2 Herbicides 

Herbicides are the major class of pesticides to control weeds. Little attention is paid to 
herbicides as a source of pollutants; mainly because with a few exception; most herbicides 
have not appreciable mammalian toxicity. Among toxic herbicides are paraquat (LD 5 o=125 
mg/kg) and dinoseb (LDso=58 mg/kg); however widely used herbicides including 2,4-D 
and glyphosate are not highly toxic to mammals. On the other hand groups of herbicides 
that have potential to persist in soil and enter surface water include triazines, sulfonylureas, 
phenylureas and uracils. Laboratory experiments have shown that among four triazines; 
prometryn and terbutylazine half-lives were 263 and 366 days in ground water respectively. 
The half lives of simazine and atrazine were shorter than prometryn and terbutylazine 
(Navarro etal. 2004). 

Sulfonylureas are high potent herbicides group effective at very low dose (10-15 g/ha), for 
that reason persistent herbicides from previously sprayed farms may damage the next crop. 
These herbicides are able to penetrate into deeper layers of the soil profile, where they have a 
relatively high persistence. A number of sulfonylureas were detected in wetland sediments. 
Etametsulforun methyl, sulfosulforun and metsulforun-methyl were determined in wetland 
sediments with mean concentration ranging from 1.2 to 10.0 jig kg- 1 (Degenhardt et al. 2010). 
According to Cessna et al (2006) a half-life of 84 days was observed for metsulforun-mathyl in 
farm dugouts. Residues of 10 herbicides were detected in prairie farm dugouts. 2,4-D was the 
most frequent with median concentration 0.05 ng L-!(Cessna & Elliot, 2004). 
Based on these studies, herbicides have different tendency for binding to soil colloids and so 
have different movement ability. 

5.3 Fungicides 

Fungicides are substances that destroy or inhibit the growth of fungi. Fungicides are used in 
agriculture and industry. Early fungicides were organic derivatives of metals such as 
mercury. Organomercury fungicides were widely used as seed dressing to control diseases 
of cereals. Although mercury content of these fungicides formulation were less than 5%, the 
main concern is the side effects of residues remaining in the environment long enough to 
enter soil and water. Both inorganic and organic compounds of mercury are toxic, however 
organic compounds are more lipophilic than inorganic and are liable to adsorption by soil 
colloids and storage in fat depot. Bioconcentration factor up to 100000 times is reported for 
the methyl mercury content in fish (USEPA, 1980). Dithiocarbamates (e.g. mancozeb, thiram, 
zineb and maneb) are the first synthetic organic fungicides. Some fungicides are toxic to 
aquatic organisms. Maneb is highly toxic to fish and triadimefon is highly toxic to 
crustaceans. Dithiocarbamate fungicides have low persistence. Among high persistent 
group of fungicides are triazoles (penconazole, myclobutanil and flusilazole), carboximides 
(boscalid) and pirimidines (fenarimol) (Wightwick et al., 2010). 

6. Conclusion 

The use of pesticides is essential for protecting agricultural products from pest damages; 
however their adverse effects are inevitable almost on all habitats. From the preceding 



Ecological Impacts of Pesticides in Agricultural Ecosystem 159 

information it is clear that side effects of pesticides on natural enemies, emergence of 
resistant populations and entrance of pesticides into the environment are the main issues 
that have been considering for a long time. More precise methods should be considered to 
evaluate these adverse impacts at the population-level and ecosystem as well as laboratory- 
based and individual-level assessment. Life table response experiments reveal total effects of 
any pesticides on an individual (target or non-target) at the population level. However, most 
publications in the field of insect toxicology are based on individual-level bioassays. 
Meanwhile, population genetics and resistance inheritance have mostly been ignored in 
insect toxicology which can provide great information on the ecological impacts of 
pesticides in agricultural ecosystems. It is believed that modern sciences such as insect 
biotechnology and nanotechnology facilitate designing novel and effective pesticides with 
less adverse effects in the environment. 

7. References 

Abbott, W.S. (1925). A Method of Comparing the Effectiveness of an Insecticide. Journal of 

Economic Entomology, Vol.18, No.2, (April 1925), pp. 265-267, ISSN 0022-0493 
Andrewartha, H. & Birch, L. (1954). The Distribution and Abundance of Animals, University of 

Chicago Press, ISBN 0226020266,Chicago, Illinois. 
Araujo, R.A., Guedes, R.N.C., Oliveira, M.G.A. & Ferreira, G.H. (2008a). Enhanced Activity 

of Carbohydrate- and Lipid-metabolizing Enzymes in Insecticide-resistant 

Populations of the Maize Weevil, Sitophilus zeamais. Bulletin of Entomological 

Research, Vol. 98, No. 4, (February 2008), pp. 417-424, ISSN 0007-4853 
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Environmental Impact and Remediation of 
Residual Lead and Arsenic Pesticides in Soil 

Eton Codling 

Agricultural Research Service, US Department of Agriculture, 

USA 



1. Introduction 

Chemical control of insects is considered one of the most beneficial developments of 
civilization (Klassen & Schwartz, 1983). As long ago as 1000 B.C., sulfur compounds were 
used to control insects in Asia Minor (National Academy of Science, 1969). However, the 
extensive use of chemicals to control pests has developed only in the last 150 years. The first 
example of large-scale effective chemical control of an insect pest occurred in 1867, with the 
use of Paris green (copper acetoarsenate) to control Colorado potato beetle, Leptinotarsa 
deecemlineata (Say). Paris green was later used to control codling moth, Laspeyresia pomonella 
(Linnaeus), on fruit trees (Klassen & Schwartz, 1983). Due to its effectiveness in controlling 
gypsy moth, Porthetria dispar (Linnaeus), lead arsenate replaced Paris green in New England 
in 1892. Lead arsenate was later used to control codling moth in apple, plum, and peach 
orchards (Klassen and Schwartz, 1983; Peryea, 1998a). 

This chapter will focus on the inorganic pesticide lead arsenate (PbHAsOzi) and its effects on 
the environment. Both lead (Pb) and arsenic (As) have been used to produce a large number 
of chemical and manufactured products. Some of these products have been used in 
agriculture as defoliants, insecticides, and fungicides to control pests in apple, plum, and 
peach orchards, turf, vegetable crops, and on cattle. From the late 1800s to about 1947, lead 
arsenate was the most commonly used insecticide for control of codling moth in deciduous 
tree fruit orchards in countries throughout the world, including the USA, Australia, Canada, 
New Zealand, England, and France, because of its low cost, high efficiency, and low 
phytotoxicity (Focus, 2006; Peryea and Kammereck, 1997 and Shepard, 1951). The wide use 
of lead arsenate significantly increased its annual production during the early 1900s. 
Worldwide, lead arsenate production increased from 2,268 metric tons in 1908 to more than 
41,000 metric tons in 1944. Even though the total amount of lead arsenate used on orchards 
is not known, this pesticide was applied frequently and at high application rates. Annual 
application rates as high as 215 kg Pb ha- 1 and 80 kg As ha- 1 were recommended for apple 
orchards (Peryea and Creger, 1994). Such high application rates helped minimize the 
development of resistant insects, a problem that farmers were facing with other insecticides. 
Moreover, the fact that lead arsenate has multiple sites of action made it unlikely that insect 
resistance could be achieved with single mutations (Georghiou, 1983). Lead arsenate was 
used as an insecticide until the introduction of the organochlorine 
dichlorodiphenyltrichloroethane (DDT) in the 1940s (Peryea 1998a; Wolz et al, 2003). 
However, lead arsenate continued to be used in some locations into the 1970s and was not 
officially banned until 1988 (Focus, 2006). 



170 Pesticides in the Modern World - Risks and Benefits 

2. Environmental effects of lead arsenate pesticides 

2.1 Occurrence of lead arsenate in the environment 

In the early 1900s, fear and concern arose about the potential for retention of excessive 
pesticide residues on fruits and vegetables treated with lead arsenate. This concern became a 
reality in 1919, when western pears were condemned in Boston because of excessive arsenic 
residues (Klassen & Schwartz, 1983). Arsenate residue was also observed in fruits grown on 
lead arsenate contaminated soils in Canada. Arsenic concentrations in fruit juice and juice 
concentrate were higher when prepared from skin and cores. Arsenic concentrations in fruit 
and fruit products were influenced by the size of fruits at spraying and by spraying 
frequency (Bishop & Chisholn, 1966). 

As a result of repeated lead arsenate application, lead and arsenic concentrations increased 
significantly in orchard soils. In one orchard soil studied, lead concentration ranged from 
500 to 1500 mg kg- 1 and arsenic concentration ranged from 200 to 500 mg kg- 1 , whereas the 
concentrations found in uncontaminated soil normally range from 2 to 300 mg kg- 1 for lead 
and 0.1 to 20 mg kg- 1 for arsenic (Alloway, 1995). In an apple orchard with 70 years of lead 
arsenate use, Frank et al. (1976) reported lead concentrations in the range of 6.4 to 774 mg 
kg- 1 and arsenic concentrations from 7.4 to 121 mg kg- 1 . The extent of contamination is 
considerable in former fruit growing areas. It has been estimated that approximately five 
percent of the soils in New Jersey are affected with lead arsenate, for example, while about 
188,000 acres in Washington State and 50,000 acres in Wisconsin are contaminated (Focus, 
2006). 

Lead and arsenic concentrations in orchards soils vary depending on the type of orchard 
(peach, plum, or apple), soil type, organic matter content, rate and frequency of pesticide 
application, and management practices after old trees are removed. Lower soil lead and 
arsenic concentrations were observed in peach orchards and vineyards than in apple 
orchards, due to the infrequency and lower rate of lead arsenate application in the former 
(Frank et al., 1976). When replanting orchards, some farmers, after removing the old trees, 
shift the rows when replanting new trees in order to protect them from lead and arsenic 
toxicity. Under this management practice, there is little disturbance of the surface, and lead 
and arsenic concentrations will be much higher in the surface soil compared to their levels 
under management practices in which fields are plowed after removal of old trees and then 
planted with agronomic crops such as corn, wheat, and soybean for two or three years 
before being replanted with new trees. Lead and arsenic concentrations in the surface soil 
under this management will be lower due to the mixing of the surface and subsurface soil. 
This practice will increase lead and arsenic in subsurface soils, which, in turn, will increase 
the potential for vertical movement of lead and arsenic to ground water, especially if these 
soils are sandy. 

Because lead and arsenic generally do not dissolve, biodegrade, or decay, are not rapidly 
absorbed by plants, and do not readily move through the soil profile, they remain in the soil 
long after their use (Wu et al., 2010). When lead arsenate reaches the soil, it undergoes 
hydrolysis, separating into lead and arsenic, which are bound to soil particles and organic 
matter and become immobilized. Lead is only slightly soluble and therefore accumulates in 
the surface soil (0-15 cm depth). Arsenic is slightly more soluble and will move through the 
soil profile (Focus, 2006). Arsenic mobility is enhanced by addition of phosphorus (Peryea 
& Kammereck, 1997). Arsenic is more mobile compared to lead regardless of the soil type 
and texture (Eflving et al., 1994). When lead is applied to soil, it will react with sulfate, 



Environmental Impact and Remediation of Residual Lead and Arsenic Pesticides in Soil 171 

phosphate, and carbonate to formed complexes such as lead sulfate (PbSQj), lead carbonate 
(Pb3 (OH)2 CO3), and chloropyromorphite (Pbs(P04)3Cl). These compounds vary in their 
solubility in the soil. Lead is also adsorbed directly to clay minerals and indirectly by 
forming complexes with organic matter (such as humic and fulvic acids) that is adsorbed 
onto soil solids (Harrison & Laxen 1984). In some cases, lead has accumulated in orchard 
soils in amounts equal to the application rate. In a survey of soils from 31 apple orchards, 
soil lead concentration averaged 821 mg kg- 1 , almost equal to the 817 mg kg- 1 applied, while 
mean soil arsenic concentration was 188 mg kg- 1 , considerably lower than the 245 mg kg- 1 
that was applied (Frank et al., 1976). 

2.2 Lead and arsenic toxicity 

Neurological impairment in children and hypertension in adults are the main health 
problems associated with chronic high lead levels in the blood. Lead toxicity in humans also 
affects red blood cells and their stem cells, the kidney, heme biosynthesis, vitamin D 
metabolism, and the neurobehavioral development of newborns, infants, and children 
(Carrington & Bolger 1992; Dudka & Miller 1999; Needlemann et al, 1990; Wolz et al, 2003). 
It has been suggested that blood Pb levels should be no higher than 6 jig dk 1 in children to 
avoid neurological symptoms and no higher than 25 jig dk 1 in adults to prevent 
hypertensive symptoms. Blood Pb levels above 10 u.g dk 1 could result in spontaneous 
abortion and potential damage to the fetus in women who are pregnant. Dietary exposure 
that results in these blood levels of concern was estimated to be 60 ng Pb per day for 
children age 6 and younger, 150 jig Pb per day for children 7 years and older, 250 jig Pb per 
day for pregnant mothers and 750 jig Pb per day for adults (Carrington & Bolger, 1992). 
In humans, circulatory disorders, skin cancer, and internal cancer are the main hazards 
related to arsenic exposure (Chaney & Ryan 1994; Dudka & Miller, 1999). Although arsenic 
is readily absorbed by humans, 40 to 70 percent of As intake is absorbed, metabolized, and 
excreted within 48 hours. The minimal risk level (MRL) for chronic oral ingestion of arsenic 
has been estimated to be 0.3 ug arsenic kg- 1 day -1 (ATSDR, 1998; Wolz et al., 2003). Arsenate 
(V) and arsenite (III) are the dominant inorganic species of arsenic in soils (Chaturvedi, 
2006). Arsenate is the predominant species in aerobic soils and arsenite is dominant under 
anaerobic conditions (Chaturvedi, 2006; Smith, 1998). Inorganic arsenic is very toxic to 
plants because phosphate and arsenate are analogs and are therefore absorbed by the same 
transport system ( Meharg, et al., 1994; Meharg, 1992). When arsenic is absorbed into the 
plant it interferes with plant metabolic processes, uncouples phosphorylation, and inhibits 
phosphate uptake, resulting in purpling of lower leaves, symptoms that are similar to those 
of phosphorus deficiency (Cox & Kovar, 2001; Gang et al., 2006). 

2.3 Risk assessment of lead arsenate exposure 
2.3.1 Introduction 

It has been reported that the health risk of living on old orchard land contaminated with 
lead arsenate is very low (Focus, 2006). Nevertheless, there is concern that when such 
contaminated land is converted to other uses such as vegetable crop production and 
residential development, (homes, schools, child care facilities, and parks) lead and arsenic 
will enter the food chain. It has been estimated that that over 6 million acres of farm land 
have been converted to non-agricultural uses (Focus, 2006). Because excess consumption of 
non-essential metals such as lead and arsenic can result in neurological, bone, and 



172 Pesticides in the Modern World - Risks and Benefits 

cardiovascular diseases, impaired renal function, and various cancers even at low levels 
(Calderon, 2000; Jarup, 2002; Khan et al., 2003, Miller et al., 2004; Watt et al., 2000), there is a 
need for the development of risk assessment of these soils. The development of any risk 
assessment for these soils must consider both toxicity and exposure (Carrington & Bolger 
1992; Pocock et al., 1984). Presently, each state has its own guidelines on the utilization of 
lead and arsenic contaminated orchard soils. However, with the increasing conversion of 
old orchard land to residential development or to other agricultural uses, a national effort is 
needed to prevent excessive human exposure to lead and arsenic. 

2.3.2 Pathways of lead and arsenic uptake 

Lead and arsenic can enter the human or animal body through direct or indirect pathways. 
A direct pathway is the unintentional consumption of lead and arsenic via the drinking of 
contaminated water or the inhalation and/or ingestion of contaminated soil and dust. An 
indirect exposure pathway is consumption of plants that have taken up Pb and/ or As from 
the soil. The direct ingestion of lead and arsenic contaminated soils, especially by children, 
is considered the most important exposure route of arsenic and lead to the human body 
(Dudka & Miller. 1999). The segment of our population most vulnerable to lead poisoning is 
children below the age of 5 years, due to their unintentional ingestion of contaminated soil 
through hand to mouth activity or by breathing house dust brought inside the house on 
shoes (Chaney & Ryan. 1994). A recent risk analysis which is considered conservative stated 
that a soil arsenic concentration of 40 mg kg -1 and a soil lead concentration of 300 mg kg- 1 do 
not result in excessive intake of arsenic and lead by humans as evaluated by the direct 
ingestion exposure mode (Dudka & Miller. 1999; US Environmental Protection Agency 
[USEPA], 1993). 

2.3.3 Plant uptake of lead and arsenic 

Even though plants do not readily take up lead and arsenic in large quantities, research has 
shown that some crops will remove lead and arsenic from lead arsenate contaminated soils. 
Plant lead concentration increased in some crops grown on lead arsenate contaminated soils, 
exceeding the 2.0 mg kg- 1 Canadian residue tolerance level (Chisholm, 1972). Miller et al, 
(2004) observed that arsenic concentrations in agricultural produce grown on lead and arsenic 
contaminated soils near a Columbia mining area were below existing Canadian guidelines for 
arsenic content in commercially sold vegetables, but that lead levels in carrots, lettuce, and 
beetroots from some locations exceeded the recommended guideline of 2.0 mg kg- 1 Pb. They 
concluded that the greatest risk was from consumption of contaminated soil particles adhering 
to vegetables, and that this risk can be reduced by washing and peeling vegetable crops before 
eating them. Carrot lead and arsenic concentrations increased when grown on lead arsenate 
contaminated soils (Zandstra & Dekryger, 2007). 

In a growth chamber study, Codling et al. (2011, in press) measured lead and arsenic uptake 
by carrots grown on five orchard soils with history of lead arsenate use, with total soil 
arsenic and lead ranging from 93 to 291 and 350 to 961 mg kg- 1 , respectively. Arsenic 
concentration in peeled carrots ranged from 0.38 to 1.64 mg kg- 1 dry weight compared to 
0.05 mg kg- 1 in the control. Lead concentration in peeled carrot ranged from 2.67 to 7.32 mg 
kg- 1 , compared to 0.19 mg kg- 1 in the control. Lead concentration was higher in peeled roots 
compared to the peel and shoot, while arsenic concentration was higher in the shoot and 
peel than in peeled roots. This study demonstrated that carrots will accumulate lead in 



Environmental Impact and Remediation of Residual Lead and Arsenic Pesticides in Soil 173 

edible tissue. Lead in food is less well absorbed by humans than lead in water, and further 
studies are needed to determine what fraction of lead and arsenic in such carrots are 
bioavailable to humans. 

There is a possibility that lead arsenate contaminated lands may be used for rice production. 
A greenhouse study was conducted to determine arsenic uptake by rice from two lead 
arsenate contaminated soils under flooded conditions (Codling, 2009). Flooding reduced 
grain yield and increased grain arsenic concentration in both soils. Lead concentration in the 
grain decreased with flooding for one of the soils but increased for the other. Lead and 
arsenic concentrations observed in the rice grain would not be expected to become a human 
health risk. However, the bioavailabilities of lead and arsenic in this rice grain need to be 
determined. Arsenic and lead concentrations in the straw and husk were much higher than 
in the grain. Straw from rice grown on these soils under flooded conditions could indirectly 
become a human health risk because rice straw is used for livestock feed and bedding. 

2.3.4 Animal uptake of lead and arsenic 

Herbivores and their predators that live in old orchards with history of lead arsenate use are 
at risk of lead and arsenic contamination (Elfving et al., 1978). Earthworms, for example, 
have been shown to concentrate lead from these soils (Ash & Lee, 1980; Morgan & Morgan, 
1999. Worm eating birds will accumulate lead from consuming these worms. Animals such 
as meadow voles have been shown to accumulate high levels of lead and arsenic in their 
liver, kidney, and bones compared to control animals (Haschck, 1979). Predators of these 
animals such as owls, hawks, and foxes potentially will accumulate lead and arsenic in their 
tissue. Animals grazing on these contaminated soils unintentionally consume large amounts 
of soil containing lead and arsenic (McGrath et al., 1982). Even though there is no evidence 
of human lead and arsenic toxicity from eating animal tissue that was grazed on lead 
arsenate contaminated soil, there is a potential for lead and arsenic entering the human food 
chain via this route. 

3. Remediation of lead arsenate contaminated soil 

3.1 Introduction 

Several remediation methods have been proposed and used for remediating lead arsenate 
contaminated soils, including removal and replacement of surface soil, chemical treatment 
in situ, establishment of a grass cover to prevent erosion and direct contact with humans, 
and phytoremediation (Peryea 1998b; Codling & Ritchie 2005). Physical removal of 
contaminated soil by excavation is acceptable and has been used. The cost of excavation, 
however, is quite expensive, with cost ranging from US$ 25,000 to US$ 1 million per acre, 
depending on the depth of soil removed, availability of a disposal site, and cost and 
availability of replacement uncontaminated soil (Peryea 1998a). This method would not be 
applicable for the large areas that are contaminated with lead arsenate. Remediation by 
dilution, such as by mixing contaminated surface soil with uncontaminated subsurface soil, 
may not be acceptable because arsenic in the subsoil may leach to the ground water. 

3.2 In situ remediation of lead and arsenic 

In situ inactivation methods reduce the hazards associated with contaminated soils through 
the use of chemicals that change the ionic and/or molecular species of metals to stabilize the 



174 Pesticides in the Modern World - Risks and Benefits 

metal chemically and physically in place (Berti & Cunningham, 1997; Brown et al v 2004). 
This method has been used effectively to sequester either lead or arsenic in soils 
contaminated from metal smelters, leaded gasoline, lead paint, lead batteries, cattle dips, 
and arsenic treated lumber. Remediation of lead arsenate contaminated soils, however, is 
more challenging because of (1) the vast amount of lead arsenate contaminated soil 
throughout the world (Peryea & Kammereck 1997), and (2) some of the common in situ 
remediation methods that have been proven effective for the remediation of lead 
contaminated soils result in the release of arsenic from lead arsenate contaminated soil, 
thereby creating a new environmental problem (Codling, 2007; Peryea 1991b). Phosphate, 
for example, has been shown to be very effective in sequestering lead in contaminated soils 
(Chaney & Ryan 1994; Ruby et al., 1994), but this remediation method is not suited for lead 
arsenate contaminated soil; because arsenate and phosphate exhibit similar physicochemical 
behavior in soil and compete directly for sorption sites on soil particles, the use of 
phosphate on a lead arsenate contaminated soil will promote arsenic release from the soil 
into the soil solution phase, threatening the ground water (Dupanport & Peryea 1991b; 
Eflving et al., 1994; Peryea & Kammereck 1997; Peryea, 1991). Increasing the number of 
adsorption sites via the addition of high oxide minerals such as iron and manganese might 
allow for the resorption of arsenate after its release caused by phosphate competition during 
lead sequestration. 

Another in situ method that has been considered for the remediation of lead arsenate 
contaminated soils is biomethylation, in which the soil is flooded after application of a 
carbon source such as apple pomace. Under the high carbon and flooded condition, 
biomethylation of arsenic will occur (Peryea, 1991b). However, the quantity of lead arsenate 
contaminated land and the topography of these sites would make biomethylation difficult 
and expensive (Focus, 2006). 

3.2.1 Long term study of in situ remediation of lead arsenate contaminated soil 

Codling (2007) determined the effect of amendment with calcium carbonate, iron, and 
phosphate on water-extractable lead and arsenic in two orchard soils with history of lead 
arsenate use. A soil from Maryland (Thurmont sandy loam) had total lead and arsenic 
concentrations of 677 and 133 mg kg 1 , respectively, and a soil from Washington State 
(Burch) had total lead and arsenic concentrations of 482 and 93 mg kg- 1 , respectively. 
Calcium carbonate, iron oxide, and phosphorus (as potassium phosphate) were mixed with 
these soils as individual treatments and as combinations. Soils and amendments were mixed 
and allowed to incubate for 60 weeks. Each treatment was sampled at 2, 4, 6, 8, 10, 16, and 60 
weeks for water-extractable lead and arsenic. Iron oxide treatment without calcium 
carbonate did not change water-extractable arsenic concentration for both soils, compared to 
the control. The phosphate and iron+phosphate treatments increased water-extractable 
arsenic compared to the control (Figure 1, only Thurmont data shown). In these treatments, 
water-extractable arsenic concentrations were higher than the recommended drinking water 
limit of 10 ng IA Application of phosphorus and iron plus phosphorus increased water- 
extractable lead concentration for both soils with and without calcium carbonate, compared 
to the control and the iron alone treatment (Figure 2, only Thurmont data with calcium 
carbonate shown). The higher water-extractable lead concentrations observed with the iron 
plus phosphorus treatment suggests that iron reacted with phosphorus making it less 
available for lead precipitation. Because of these observed increases in water-extractable 
lead and arsenic caused by application of iron and phosphorus to lead arsenate 



Environmental Impact and Remediation of Residual Lead and Arsenic Pesticides in Soil 



175 



contaminated soils, this in situ remediation method should not be use without further 
studies to determine the appropriate ratio of iron to phosphorus needed to sequester lead 
and arsenic in these soils. 



3.3 Phytoremediation of lead arsenate contaminated soil 

Using plants to remove toxic metals from soils, a process known as phytoremediation, is an 
inexpensive alternative to conventional methods (Lim et al 2004). In order for a plant to be 
considered a hyperaccumulator, it must accumulate at least 1000 mg kg- 1 of the metal in the 
above ground tissue. Chaney et al. (1994) demonstrated that Alyssum murale Waldst. & Kit. 
will accumulate nickel from high nickel serpentine soil. Francesconi et al. (2002) and Ma et 
al. (2001) demonstrated that Chinese brake fern (Pteris vittata L) and silver fern Pityrogramma 
calomelanos (L) can hyperaccumulate arsenic. Some researchers do not believe 
phytoremediation of lead arsenate contaminated soil holds much promise because it is slow, 
potentially taking decades or longer to effectively remove the contaminant. While other 
researchers believe that phytoremediation is a cost effective, non-intrusive technology that 
needs improvement (Alkorta, et al., 2004). Alkorta, et al. (2004) stated that improvement of 
the capacity of plants to tolerate and accumulate metals by genetic engineering should open 
up new possibility for phytoremediation. They also suggested that a better understanding of 
metal uptake and translocation mechanisms and the external effects of phytoremediation 
should also increase its application. Research is ongoing to identify plants that could 
potentially be used as accumulators of lead and/ or arsenic on lead arsenate contaminated 
soils. In a greenhouse study, Codling & Ritchie (2005) tested Eastern gamagrass [Tipsacum 
dactyloides (L.)] for lead and/or arsenic accumulation from two lead arsenate contaminated 
orchard soils. This species was chosen because the plant has an extensive root system and is 
used to reduce soil erosion on disturbed soils. Eastern gamagrass did not remove substantial 
amount of arsenic from these soils, making this species a poor candidate for 
phytoremediation of lead arsenate contaminated soils. 




10 



Incubation Time (Weeks) 

Fig. 1. Water-extractable arsenic in limed iron- and phosphorus-amended lead arsenate 
contaminated Thurmont soil during incubation at 26 °C. Values are mean and standard 
deviation (n=3). 



176 



Pesticides in the Modern World - Risks and Benefits 



E 

to 30 












A 


— •— + L control 


' 


-o- +L +Fe 
-^T- +L +P 




' 




— a- +L +Fe +P 




■ 




d VT— — ? 




0— 





Incubation Time (Weeks) 

Fig. 2. Water-extractable lead in limed iron- and phosphorus-amended lead arsenate 
contaminated Thurmont soil during incubation at 26 °C. Values are mean and standard 
deviation (n=3). 

4. Conclusions 

The existence of large areas of lead arsenate contaminated orchard soils and their increasing 
conversion to vegetable crop production and to residential development has created a 
potential risk to public health. Young children are especially at risk because of their 
unintentional consumption of soil. Children exposed to lead may develop neurobehavioral 
impairment, while arsenic is a human carcinogen. Humans can also be exposed indirectly to 
lead and arsenic through the consumption of vegetable crops grown on contaminated soils, 
although the bioavailability of lead and arsenic in vegetable crops consumed by human and 
animals is not known. Because hundreds of thousands of acres have been contaminated 
with lead arsenate, removal of contaminated soil and replacement with clean surface soil is 
not economically feasible. Chemical in situ treatment with phosphorus, although effective in 
sequestering soil lead from other sources, has been shown to increase the leaching of arsenic 
to ground water in lead arsenate contaminated soils. Application of iron oxide has been 
shown to be effective in sequestering arsenic in lead arsenate contaminated orchard soils. 
Using plants to remove metals from contaminated soil (phytoremediation) is a method that 
is being considered for removing lead and arsenic from soils, but even if lead and arsenic 
accumulating plants are identified, this method may be too slow to be practical. Further 
research needs to be done on remediation of lead arsenate contaminated soils. Presently, 
each state has its own guidelines on the utilization of lead and arsenic contaminated orchard 
soils. However, with the increasing conversion of old orchard land to residential 
development or to other agricultural uses, a national effort is needed to prevent excessive 
human exposure to lead and arsenic. 



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Environmental Impact and Remediation of Residual Lead and Arsenic Pesticides in Soil 177 

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Soils. 2^ Ed. pp. 354. Chapman & Hall New York N Y. 
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Agency for Toxic Substances Disease Register (ATSDR), 1998. Toxicological profile arsenic 

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Berti, W. R. and Cunningham S. D. 1997. In place inactivation of lead in lead contaminated 

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Bishop, R. F. and Chisholm, D. 1966. Arsenic spray residues on apples and in some apple 

products. Canadian Journal of plant Science 46: 225-231. 
Brown, S., Chaney, R., Hallfrisch, J., Ryan, J. A. and Berti, W. R. 2004. In situ soil treatments 

to reduce the phyto and bioavailabilityof lead, zinc and cadmium. Journal 

Environmental. Quality. 33: 522-531. 
Calderon, R. L. 2000. The epidemiology of chemical contaminants of drinking water. Food 

Chemistry and Toxicology. 38: S 13-S20. 
Carrington, C. D., and Bolger P. M. 1992.An assessment of the hazards of lead in food. 

Regulatory Toxicology and Pharmacology. 16: 265-272. 
Chaney, R. L. and Ryan, }. A. 1 994. Risk based standards for arsenic, lead and cadmium in 

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Chaney, R.L., Malik, M., LI, Y. M., Brown, S. 1., Brewer, E. P., Angle, J. S. and Baker, A. J. 

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Chaturvedi, I. 2006. Effect of arsenic concentrations and forms on growth and arsenicuptake 

and accumulation by Indian mustard (Brassica Juncea L.) genotypes. Journal of 

Central European Agriculture 7:31-40. 
Chisholm, D. 1972. Lead arsenate and copper content of crops grown on lead arsenate 

treated soils. Canadian Journal of Plant Science. 52: 583-588. 
Codling, E. E., Chaney, R. L. and Green. C. E. 2011. Accumulation of lead and arsenic by 

carrots grown on four lead-arsenate contaminated orchard soils. Submitted to 

Journal of Environmental Quality. 
Codling, E. E. 2009. Effect of flooding lead arsenate-contaminated orchard soil on growth 

and arsenic and lead accumulation in rice. Communications in Soil Science and Plant 

Analysis. 40: 2800-2815. 
Codling, E. E. 2007. Long term effects of lime, phosphorus, and iron amendments on water 

extractable arsenic, lead and Bioaccessible lead from contaminated orchard soils. 

Soil Science. 172: 811-819. 
Codling, E. E. and Ritchie, J. C. 2005. Eastern gamagrass uptake of lead and arsenic from 

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170: 413-424. 
Cox. M. S. and. Kovar, J. L. 2001. Soil Arsenic effects on canola seedling and growth and ion 

uptake. Communications Soil Science and Plant Analysis. 31:107-117. 
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in soilcontominated with lead arsenate. Water Air and Soil Pollution. 57-58:101-110. 
Dudka, S. and Miller, W. P. 1999. Permissible concentrations of arsenic and lead in soils 

based on risk assessment. Water Air and Soil Pollution. 113: 127-132. 



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Eflving, D. C, Wilson, K. R., Ebel, J. G. Jr., Manzell, K.L.,Gutenmann,W. H., and Lisk, D. J. 

1994. Migration of lead and arsenic in old orchard soils in the Georgan Bay region 

of Ontario. Chemosphere, 29: 2. 407-413 
Eflving, D. C, Haschek, W. M., Stehn, R. A., Bache, C. A., Lisk. D. J. 1978. Heavy metal 

residues in plants cultivated on and in small mammals indigenous to old orchard 

soils. Archives of Environmental Health. 33: 95-99. 
Francesconi, K., Visoottiviseth, P., Sridokchan, W. and Goessler, W. 2002. Arsenic species in 

an arsenic hyoeraccumulating fern, Pityrogramma colomelanos: A potential 

phytoremediator of arsenic-contaminated soils. Science of the Total Environment. 

284:27-35. 
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pesticide residue in orchard soils and vineyards of southern Ontario. Canadian 

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Focus, 2006. The apple bites back: Cleaning old orchard orchards for residential 

development. Environmental Health Perspectives. 114: pp. 1-8. Retrieved from 

http://ehp.niehs.nih. gov/members/2006/114— 8/focus.html 
Gang, C. N, Zhu, Y. G, Tong, Y. P., Smith, S. E. and Smith, F. A. 2005. Arsenic uptake and 

distribution in two cultivars of winter wheat (Triticum aestivum L). Chemosphere. 

62: 608-615. 
Georghiou, G. P. 1980. Insecticide resistance and prospects for its management. Residue 

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Harrison, R. M. and Laxen, D. P. H. 1984. Lead in soils. In Lead pollution cause and control. 

Methuen , inc. New York,. Pp. 55-69 
Haschek, W. M., Lisk, D. J. Stehn, R. A. 1979. Accumulation of lead in rodents from two 

orchard sites in New York in Animals as monitors of environmental pollution, 

National Academy of Science, Washington, DC. pp. 192-199. 
Jarup, L. 2002. Cadminum overload and toxicity. Nephrol Dial Transpl. 17 : 35-39. 
Jones, F. T. 2007. A broad view of arsenic. Poultry Science 86: 2-14. 
Khan, M. M. H, Sakauchi, F., Sonoda, T., Washio, M. and Mori, M. 2003. Magnitude of 

arsenic in tube-well drinking water in Bangladesh and its adverse effects on human 

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hyperaccumulates arsenic. Nature. 409: 579. 
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sheep. Irish Journal of Agriculture Research. 21: 135-145. 
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42. 



10 

Arsenic - Pesticides with 
an Ambivalent Character 

Arna Shab and Catharina Crofimann 

Clinic for Dermatology, Venerology and Allergology Wiesbaden, 

Germany 



1. Introduction 



Arsenic (As) is a naturally occurring ubiquitous element. It is found in the environment in 
the earth crust and quantities in media such as soil, water, rock and air. It is present in the 
environment naturally and due to human activities and other industrial processes such as 
mining and coal-fired power plants. Arsenic has also been used as a pesticide to protect 
animals, wood, fruit and vegetables from insects. Because of its therapeutic properties, 
arsenic has also been used as a medicinal agent. The dark side of the medicine was the 
reputation as an attractive poison 

Arsenic is mainly transported in the environment by food, except in areas with high levels 
of arsenic in the drinking water e.g. India, Taiwan and Bangladesh (2004; Singh, Kumar, and 
Sahu 2007). The outbreak of As was triggered by deep drilled wells and the desire to obtain 
microorganism-free safety drinking water. Arsenic contamination of drinking-water is a 
hazard to human health. Because of the toxicities and side effects of arsenic compounds it is 
known as a major environmental pollutant. The IARC classified arsenic and arsenic 
compounds as a human carcinogen (Group 1) (2004). But although arsenic compounds have 
been known and used for centuries, their mechanisms of interaction in humans are not fully 
elucidated. 

The paradox of arsenic compounds is that, on the one hand, they are considered extremely 
dangerous for human health with acute and chronic adverse health effects. Long-term 
arsenic exposure can lead to several types of cancer. The exposure to As has been associated 
also with non-carcinogenic effects e.g. such diabetes and cardiovascular diseases. On the 
other hand arsenic compounds are regarded as potential drugs against cancer and ranging 
from the use as poisons to applications in semiconductors and pesticides. Especially the 
discovery of organoarsenicals for the treatment of hematological malignancies and solid 
tumor has awakened interest. 

2. Background and basics 

Arsenic is a chemical element in the period table that has the symbol "As", the atomic number 
33 and an atomic mass of 74,92159 g/mol. Arsenic exhibits both metallic and non-metallic 
properties. Arsenic exists as unstable oxides and sulfides or as arsenites or arsenates of 
sodium, calcium and potassium. Arsenic has two biologically important oxidation states: 
arsenite (the trivalent form, As III) and arsenate (the pentavalent form, As V). As III is 60 times 



182 



Pesticides in the Modern World - Risks and Benefits 



more toxic than As V (Yousef, El-Demerdash, and Radwan 2008). From biological and 
toxicological view, arsenic compounds can be classified into three major groups: Inorganic 
arsenic compounds, organic arsenic compounds and arsine gas (Hardman et al. 1996). The 
metalloid is found mostly as yellow complex sulfides. Organic arsenic is non-toxic whereas 
inorganic arsenic is toxic. The inorganic forms of arsenic are yellow (AS2S3, orpiment), red 
(AS2S2 realgar) and grey to silver white (FeAsS, arsenopyrite) (Waxman and Anderson 2001). 




a) Orpiment 



BH b) Realgar 



c) Arsenopyrite 



Fig. 1. Orpiment, realgar and arsenopyrite - from left to right- (Photographs from the Ohio 
State University Newark) 



Arsenic - Pesticides with an Ambivalent Character 



183 



Arsenic compounds have no smell or taste, but heat can cause As to sublimate to gas with a 
distinctive garlic odor (Jones 2007). 

Arsenic naturally occures in the earth's surface, mostly in inorganic form (Hine, Pinto, and 
Nelson 1977). It exists in low concentrations in many rock types but is frequently associated 
with metal ore deposits e.g. Au (gold) , Ag (silver), Cu (copper) and Fe (iron) (Gochfeld 
1997). The most important natural sources of arsenic in the environment are volcanoes. The 
organic form result when arsenic combines with carbon and hydrogen. 



Compound 


CAS No. 


Molecular formula 


Arsenic 


7440-38-2 


As 


Arsenic trioxide 


1327-53-3 


As 2 3 


Arsenic pentoxide 


1303-28-2 


AS2O5 


Arsenic sulphide 


1303-33-9 


As 2 S 3 


Dimethylarsinic acid (DMA) 


75-60-5 


(CH 3 ) 2 AsO(OH) 


Potassium arsenate 


7784-41-0 


KH 2 As0 4 


Potassium arsenite 


10124-50-2 


Kas0 2 HAs0 2 



Table 1. Physiochemical properties (1980) 



3. History 

Arsenic and arsenic compounds are known since the ancient times. As early as 500 B.C. the 
ancients knew about arsenic, whose name comes from the Greek word "arsenikon" for 
potent or bold, which means orpiment form Latin auripigmentum. In the 16 th an 17 th 
centuries, red and white arsenic were put into amulets that were worn around the neck and 
close to the heart to ward off the plaques (Cullen 2008). Most arsenic is found in conjunction 
with sulfur in minerals such as arsenopyrite (AsFeS). Because of the association with ore 
and the stability of As in form like AsFeS, As was used as a "pathfinder element" in 
geochemical exploration for gold (Jones 2007). 




Fig. 2. Acidum Arsenicosum Anhydricum bottle, Global Antiques 



184 



Pesticides in the Modern World - Risks and Benefits 



Through the centuries Arsenic was a common method of homicide. The death of the French 
emperor Napoleon Bonaparte, on 5 May 1821 was believed to be a victim after drinking 
arsenic-tainted wine that was served him (Leslie and Smith 1978; Lin, Alber, and 
Henkelmann 2004). Arsenic was a popular murder weapon because of the odorless and 
tasteless properties and the poisoning result in symptoms that can be confused with other 
natural disorders. In the Middle ages arsenic was a favorite poison and has been called the 
Poison of Kings and the King of Poison (Vahidnia, van, V, and de Wolff 2007). 
Arsenic was also used as healing agents. The Greek physicians such as Hippocrates and 
Galen popularized it use for treating skin ulcers and tumors such as superficial 
epitheliomas. Arsenic has been used as topical pastes, as vapor inhalation, intravenous 
injection, orally in liquid or in solid form. A paste of the sulfides were used for treatment of 
neuralgia, rheumatism, arthritis and skin disease (Shen et al. 1997). Also Fowler's solution, a 
1% arsenic trioxide preparation, was widely used during the 19th century. Fowler's original 
recipe was described as "64 grains arsenic oxide, 64 grains purest vegetable alkali, distilled 
water half pound. Heat until clear. Cool. Add half pound spirit of lavender and make up to 
15 oz with water." (Cullen 2008). 




Fig. 3. Fowler's Solution made by Wisconsin Pharmacal Co 

It was used to treat diseases like leukemia, Hodkin's disease and pernicious anemia. 
The first organic arsenical used therapeutically was Salvarsan, which was developed by 
Paul Ehrlich 1907. It was used to treat syphilis, until penicillin became available in the 1940s. 
A model representation from Salvarsan and a picture of Ehrlich adorned the 200 
Deutschmark banknote. 



AD4829120N7 ¥ 







Fig. 4. German 200 Deutschmark banknote with hologram and Paul Ehrlich 



Arsenic - Pesticides with an Ambivalent Character 1 85 

For centuries arsenic has been used for different purposes. Arsenic was an ingredient of a lot of 
consumer products e.g. wallpapers, toys, food wrappers, cosmetics, pigments in paints - 
known as "Paris green". William Withering, an english doctor, who discovered 1775 digitalis 
was a proponent of therapies with arsenic. He argued:" Poison in small doses are the best 
medicines; and the best medicines in too large doses are poisonous (Aronson 1994). 
Arsenic -containing compounds have been used for cancer-treatment in both tradition 
Western and Chinese medicine. The first use of arsenic in the treatment of leukemia was in 
1865 by Lissauer (Lissauer and H. 1865). With the development of modern medicine against 
cancer the use of arsenic in the western world diminished. 

4. Epidemiology 

Arsenic exposure occurs from inhalation, absorption through the skin and by ingestion. 
Arsenic is mainly transported in the environment by food, which contains both organic and 
inorganic As, but mostly accrue as relatively no-toxic organic compounds (arsenobentaine 
and arsenocholine). Seafood, fish and algae are the richest organic sources (Edmonds and 
Francesconi 1987). The following table shows an overview of the arsenic content of various 
foods. 



Food 


Estimates of daily intake (nj^d) 


Milk 


1,39 


Fruits and vegetable 


0,46 


Meat 


2,14 


Cereals and bakery wares 


6,57 


Fish 


34,9 


Eggs 


0,13 


Sweeteners 


0,2 


Beverages 


4,67 



Table 2. Estimated of daily arsenic intake from diet (Sorvari et al. 2007) 

Concentrations of arsenic vary in the environment, e.g. 0,03-025 ppm in soil, 0,023-0,35 ppm 
in plants, up to 55 ppm in groundwater, 0,0001-0,08 ppm in seawater, 4-170 ppm in fish, 
0,008-0,85 ppm in wine and up to 0,00049 or 0,63 mg/m 3 in urban air (2004; Jones 2007; 
Rahman 2006; Basu et al. 2001). Contamination of arsenic in ground water is a global 
problem and millions of people are at a risk of arsenicosis. People from countries in Asia 
(Taiwan, Bangladesh, West Bengal (India) and South America (Chile and Cordoba) get 
presented to inorganic arsenic in ground water with very high concentration. The arsenic 
poisoning from drinking As-contaminated underground water was often triggered by the 
introduction of deep tube-pump wells to replace surface water. The World health 
organization (WHO) and US environment protection agency (EPA) had set up the standard 
for drinking water known as maximum contamination level (MCL) which is 10 ng/1 
(Effelsberg 1992). The WHO recommended 0.01 mg/1 of arsenic in drinking water as an 
allowable ranger for human consumption. Millions of people are compelled to use the 
drinking water higher arsenic level than MCL worldwide. In addition there are industrial 
exposures for workers, e.g. semiconductor workers and famers handle with arsenical 
herbicides. Arsenic has been used as feed additives e.g. poultry feeds. 

It was found an increase in the prevalence of skin lesions at 0,005 mg As/1 in the drinking 
water, which is a lower level than the drinking water quality standard of WHO (Yoshida, 



186 Pesticides in the Modern World - Risks and Benefits 

Yamauchi, and Fan 2004). The skin is very sensitive to As and skin lesions, which are As- 
induced, are the early effects to chronic As exposure. 

Arsenic is released to the atmosphere from both natural and anthropogenic sources. 
Tobacco smoke may contain arsenic, especially when the plants have been treated with 
arsenate insecticide. 



Country 


Daily dietary intake of total arsenic from diet (n^d) 


Bangladesh 


515 


Japan 


182 


USA 


20-130 


Spain 


245 


French 


62 


Germany 


52 


UK 


66 


Denmark 


64 



Table 3. Estimated daily intake of arsenic by the general population ((Devesa et al. 2001; 
Jelinek and Corneliussen 1977; Leblanc et al. 2005; Mohri, Hisanaga, and Ishinishi 1990; 
Saipan and Ruangwises 2009; Sorvari et al, 2007; Watanabe et al. 2004) 

The principal natural source is volcanic activity, with minor contribution by exudates from 
vegetation and wind-blow dust. Man-made emissions to air arise from the smelting of 
metals, the combustion of fuels, especially of low-grade brown coal, and the use of 
pesticides. Because of the use of numerous arsenical pesticides the arsenic concentration 
raised in the soil. 

5. Effects on human 

The biological activity of arsenic in the body covers a broad spectrum from toxic to 
therapeutic agent. Not to forget- Arsenic is a human carcinogen (IARC 2004). A number of 
studies show that arsenic is an essential element for humans. Other studies have attempted 
to show that arsenic has not been demonstrated to be essential to humans (Ohtake 2000). 
The major routes of arsenic absorption in the general population are ingestion and 
inhalation. Meat, fish and poultry account for 80 % of dietary arsenic intake (Edmonds and 
Francesconi 1987). Arsenic is absorbed in the small intestine by an electrogenic process 
involving a proton gradient. The absorbed arsenic undergoes hepatic biomethylation. The 
products are less toxic but not completely innocuous. About 50 % of the ingested dose may 
be eliminated in the urine in 3-5 days. Metabolism of As involves reduction of As V to a 
trivalent state and subsequent oxidative methylation. 

5.1 Acute effects 

After acute poisoning studies show that the highest concentration of arsenic is in the kidney 
and liver (Benramdane et al. 1999). Most cases of acute arsenic poisoning occur from accidental 
ingestion of insecticides or pesticides. Acute exposure to arsenic arise symptoms like 
abdominal pain, vomiting, diarrhea. The abdominal pain may mimic an acute abdomen 
(Mueller and Benowitz 1989). Other clinical features are muscular weakness and cramping, 
erythematous skin eruptions like diffuse skin rash and swelling of acrals. A progressive 
deterioration in the motor and sensory responses and toxic cardiomyopathy may also result 



Arsenic - Pesticides with an Ambivalent Character 



187 



leading to shock and death. Depending on the quantity of arsenic, death usually occurs within 
1-5 days. In acute poisoning the best indicator of recent ingestion (1-2 days) is urinary arsenic 
concentration. Dimethylarsinic acid is the dominant urinary metabolite compared with 
monomethylarsoonic acid (Hopenhayn-Rich, Smith, and Goeden 1993). 

5.2 Chronic effects 

Chronic ingestion of inorganic arsenic causes multisystem adverse health effects. The 
clinical features of chronic arsenic toxicity vary between individuals, population groups and 
geographic areas. In chronic arsenic ingestion, arsenic accumulates in the liver, kidneys, 
heart and lungs and smaller amounts in the gastrointestinal tract, spleen and muscles 
(Benramdane et al., 1999). High doses of arsenic cause characteristic skin manifestation, 
vascular, renal and neurological diseases, cardiovascular and chronic lung diseases and 
cancer of skin, lungs, liver, kidney and bladder. After about two weeks arsenic is deposited 
in the hair and nails. Levels between 0,1 and 0,5 mg/kg on a hair sample indicate chronic 
poisoning. Various epidemiological studies have reported that arsenic exposure is 
associated with hypertension, atherosclerosis and endothelial dysfunction (Yang et al. 2007) 
(Chen et al. 2007) (Kwok et al. 2007). Increasing exposure of arsenic is also associated with 
non insulin dependent diabetes mellitus (Wang et al. 2003). Studies reported that arsenic is 
associated with the growth retardation in children (Wang et al. 2007). 



5.3 Skin symptoms 

Skin manifestation is the most common and initial sign of chronic arsenic exposure. Chronic 
ingestion of arsenic causes characteristic melanosis, keratosis, basal cell carcinoma and 
squamous cell carcinoma (Maloney 1996). Melanosis includes hyperpigmentation, spotted 
pigmentation, depigmentation and leucomelanosis. Keratosis is a late feature of arsenical- 
dermatosis and appears especially on palm as a uniform thickening or as discrete nodules 
(Wong, Tan, and Goh 1998b). Both palmar and solar keratosis are significant diagnostic 
criterion. Bowen's disease is a precancerous lesion and predisposed to an increased 
incidence of the squamous cell carcinoma. Chronic ingestion of arsenic lead to accumulate in 
keratin rich areas of body and appears as white lines in the nails, called Mee's lines (Fincher 
and Koerker 1987). The latency period of skin lesions of arsenic after first exposure varies 




Fig. 5. Patient with plantar keratosis (2004; Wong, Tan, and Goh 1998c). 



188 



Pesticides in the Modern World - Risks and Benefits 




Fig. 6. Blackfoot diseases after chronic arsenic exposure (Better Life Laboratories, USA) 

from 20 to 50 years (Haque et al. 2003). It is described that the latent period after exposure 
can be as long as 60 years, which has been reported in patients treated with Fowler's 
solution, in vineyard workers using arsenical pesticides and from drinking contaminated 
wine (Everall and Dowd 1978). 

Many different systems within the body are affected by chronic exposure. Some of these 
systems and their associated toxic effects from chronic arsenic exposure are listed in the 
following table. 



System 


Effect and symptoms 


Skin 


Skin lesions (melanosis, keratosis) 


Cardiovascular 


Blackfoot disease, atherosclerosis, hypertension 


Hepatic 


Hepatomegaly, fibrosis, cirrhosis, altered heme metabolism 


Hematological 


Bone marrow depression (anemia, leucopenia, 
thrombocytopenia) 


Endocrine 


Diabetes 


Renal 


Tubule degeneration, papillary and cortical necrosis 


Nervous 


Peripher and central neuropathy, encephalopathy 


respiratory 


Pulmonary insufficiency, emphysem 


Gastrointestinal 


Hemorrhage 



Table 4. Human effects after chronic arsenic exposure (Singh, Kumar, and Sahu 2007; 
Schuhmacher-Wolz et al. 2009; Hughes 2002; Balakumar and Kaur 2009; Rahman, Ng, and 
Naidu 2009). 



Arsenic - Pesticides with an Ambivalent Character 1 89 

6. Toxicity 

Arsenic compounds or arsenic-containing compounds vary in toxicity to mammalian cells. 
Arsenic does not directly react with DNA or cause gene mutations, except to a small extent 
at high dose. As can cause gene amplification and chromosomal damage at lower doses and 
can enhance mutagenesis by other agents, apparently by inhibiting DNA repair. The 
following table gives an overview over the modes of carcinogenic action of arsenic. 



Modes of carcinogenic action of arsenic 



Genotoxicify 



Oxidative damage 



Modification of cell signalling 



Influence on DNA repair 



Influence on DNA methylation 



Changes in cell proliferation 



Co-mutagenesis and transformation 



Tumor promotion 



Table 5. Modes of carcinogenic action of arsenic (Schuhmacher-Wolz, Dieter, Klein, and 
Schneider 2009; Hughes 2002). 

The binding with sulfhydryl groups by arsenite compounds has the potential to influence a 
wide range of metabolic activities. Arsenic toxicity inactivates up to 200 enzymes. The 
effects of As occur through indirect alteration of gene expression via disruption of DNA 
methylation, inhibition of DNA repair, oxidative stress, or altered modulation of signal 
transduction pathways. Another indirect mechanism is the influence of growth-stimulating 
chemicals or cytokinesed generated in response to arsenic exposure. Biotranformation is the 
major metabolic pathway for inorganic arsenic in humans. Toxic inorganic arsenic species 
can be biomethylated by bacteria, algae, fungi and humans. The high affinity of arsenic for 
sulphydryl groups makes keratin-rich cells a target for arsenic. 
The order of toxicity of arsenicals is: 

Monomethylarsonic acid (MM A III) > Arsenite (III) > Arsenate (V) > MMA(V) (Singh, 
Kumar, and Sahu 2007). 

In arsenic biotransformation the intermediate product MMA III is highly toxic than other 
arsenical, which might be responsible for the arsenic-induced carcinogenesis and other 
effects (Styblo et al. 2000). As III binds thiol or sulfhydryl groups in tissue proteins of the 
liver, lungs, kidney, spleen, gastrointestinal mucosa and keratin-rich-issues (skin, hair, 
nails). By binding a wide range of metabolic activities are influenced including cellular 
glucose uptake, gluconeogenesis and fatty acid oxidation (Jones 2007). Many other toxic 
effects of arsenic compounds are detailed by Abernathy et al in 1999 (Abernathy et al. 1999). 
The acute toxicity is related to its chemical form and oxidation state. In the human adult the 
lethal range of inorganic arsenic is estimated at a dose of 1-3 mg As / kg (Schoolmeester and 
White 1980). The characteristics of acute arsenic toxicity in humans include gastrointestinal 
discomfort, vomiting, diarrhea, bloody urine, anuria, shock, convulsions, coma and death. 

7. Pharmaceutical use 

Arsenic has been used therapeutically for over 2000 years. During the 18 lh - 20 th centuries 
arsenic compounds have been used as medicines, including arsphenamine and arsenic 



190 



Pesticides in the Modern World - Risks and Benefits 



trioxide. In 1910, Paul Ehrlich introduced the arsenic-based drug Salvarsan (arsenobenzol) 
as a remedy for syphilis in all stages, a sexually transmitted disease. It was efficient in 
various similar diseases such as relapsing fever, Vincent's angina. 

Arsenic trioxide is also known as an anti-bacterial and anti-cancer agent (Bardos, tta-Gupta, 
and Hebborn 1966). Inorganic As has been also used pharmacologically for the treatment of 
eczema, pemphigus and psoriasis under the name of Fowler's solution. It was a 1 % solution 
of potassium arsenite, colored with a tincture of lavender-which contained a very high 
concentration of arsenic (Rahman 2006). Some arsenic containing drugs are still presently 
used to treat diseases like asthma rheumatism, cough, pruritus and itching (Ko 1999; Wong, 
Tan, and Goh 1998a). 

In 2000, the US Food and Drug Administration approved the use of arsenic trioxide for 
treatment of relapsed or refractory acute promyelocytic leukemia (APL), a subtype of acute 
myeloid leukemia (AML) (Antman 2001). It is based on its mechanism as an inducer of 
apoptosis (programmed cell death) (Soignet et al. 1998). 



FML-RARot degradation 



All-frans- 
retinoic acid 




Myelocyte 



Metamyelocyte Granulocyte 






pml in PODS 



Differentiation 



PML-HAPfi 



RAR (wild-type) 

- Cytoplasm 

- Nucleus 
■DNA 




PML-RARu degradation 



Myelocyte 



Arsenic trioxide 




Programmed 
cell death 



PML degradation 



Fig. 7. Effects of all-trans-retinoic acid and arsenic trioxide in the blast cells of acute 
promyelocytic leukemia (APL) (Look 1998). 

Traditional medicine products contain arsenic sulfides (realgar) and are available as pills 
and tablets. They are still used for psoriasis, syphilis, asthma, rheumatism, hemorrhoids, 
cough and pruritus and are rescribed as a health tonic, an analgesic, anti-inflammatory 
agent (Ko 1999; Wong, Tan, and Goh 1998d). In Korea arsenic is prescribed in herbal 
medicine for anal suffering such haemorrhoids (Mitchell-Heggs, Conway, and Cassar 1990). 



Arsenic - Pesticides with an Ambivalent Character 



191 



8. Industrial use 

In industry, arsenic is used to manufacture polants, fungicides, insecticides, pesticides, 
herbicides, wood preservatives, and cotton desiccants. 

In most local hardware stores arsenic-containing herbicides are readily available, the most 
common are e.g. Disodium methylarsonate (DSMO), monosodium methylarsonate (MSMA), 
monomethyl arsenic acid (MMA(V)). These compounds can kill crabgrass and other 
unwanted grass types. Arsenic trioxide (AS2O3) is commonly used as an antisecticide. 
Arsenic acid and arsenous acid are common rodenticides. The major use for arsenic is in the 
form of chromated copper arsenate, which reduces termites and ants from wood. 
Arsenic is used industrially as an additive to glass to reduce coloring, in semiconductors, in 
pigments such as Paris green (CuHAs03) and in pesticides. Paris Green is a common name 
for copper (II) acetoarsenite, which is a toxic emerald-green crystalline powder. Other 
names for the chemical are Emerald Green, Vienna Green, Schweinfurt Green and Parrot 
Green. The use has been abandoned around 1960. The III-V semiconductors are very 
important in the fabrication of LED's, tunnel diodes, infrared emitters, laser window and 
Hall-effect devices. 




Fig. 8. Paris Green bottle 
(http://theodoregray.com/periodictable/Elements/033/index.s7.html). 



192 



Pesticides in the Modern World - Risks and Benefits 



9. Arsenic, wine and profession 

Arsenic was used in vineyards for only some years as a pesticide. It was officially 
introduced as a pesticide in viniculture in 1925. Its purpose was to protect the wine plants. 
But it was banned in 1942. It was used in Germany until the mid 1950ties (Shab, 2009). 
Consumption of the so-called wine-grower's house drink led to severe symptoms and 
illnesses, especially liver damage. This homemade wine was produced by watering down 
the wine obtained from a second pressing of the grape skins. It was consumed in large 
quantities, which had a low alcohol content, from 3-5 %, but high arsenic content (Kunz and 
Kunz 2008). Exposure to arsenic has been reported to lead to cirrhosis and to angiosarcoma 
among famers exposed to arsenical insecticides. Chronic liver disease which can be caused 
by arsenic toxicity includes also steatosis and noncirrhotic portal hypertension (Von Hyman 
J.Zimmerman. 1999). 

Not only at the vineyards there was an occupational exposure to arsenic. Other important 
occupational exposure opportunities exist for processing of metal ores, roasting of pyrites in 
the chemical industry, the production and use of arsenic colors and tints for glass, porcelain 
and ceramics industry, pesticides and wood preservatives as well as at the battery and 
semiconductor. Occupational diseases caused by arsenic and its compound can be 
recognized as an occupational disease (BK-Nr. 1108 in Germany) (1964). 




I Hi ''/t *•< 






Fig. 9. Wine glass with wine yard (Markus Ebert - photographer Heidelberg/ Potsdam) 



10. Conclusion 

From history to the present, the story of arsenic is double-edged: a poisonous edge and a 
medicinal edge. Arsenic has been mentioned mainly as a poison and public health problem 



Arsenic - Pesticides with an Ambivalent Character 1 93 

than as an effective anticancer drug. Arsenic is one of the most toxic metals derived from the 
natural environment. Inorganic Arsenic is a human carcinogen, but nowadays also acts as a 
beneficial chemotherapeutic agent. The major cause of human arsenic toxicity is from 
contamination of drinking water and from As-contaminated food through fertilization. 
Current uses of arsenic compounds are in the glass industry, as a wood preservative and in 
the production of semiconductor. Over the centuries, arsenic has been used for a variety of 
purposes. In industry arsenic is used as a potential weapon against insecticides concerning 
humans as a modern weapon. Arsenic compounds became available e.g. in Fowler's 
solution as indication for skin conditions and treatment for acute and chronic diseases. 
Arsenic affects many cellular and physiological pathways, which is useful in treating 
malignancies like hematological cancer and solid tumors. The ability of arsenic trioxide to 
treat APL has changed the point of view. 

Still today moderately elevated concentrations of inorganic arsenic in drinking water is a 
major public health concern as well as arsenic exposure from food, especially rice products 
(Sun etal. 2008). 

Chronic arsenicism may lead to multiple benign skin diseases as well as potentially fatal 
skin and visceral malignancies e.g. lungs, bladder, liver kidneys. Pigmentation changes and 
hyperkeratosis are the earliest signs of toxicity from chronic exposure. People with chronic 
arsenicism should undergo regular skin and systemic examination. There are no evidence 
based treatments to reduce chronic arsenic poisoning, but antioxidants have been 
advocated: Pharmacological interventions such as vitamin C, folic acid, vitamin bl2 have 
been identified to halt the development of arsenic-induced toxicity. More studies are 
needed. The essential and basic efforts for the reduction of chronic arsenic toxicity are 
prevention. Although current exposure to arsenic is decreasing, continual surveillance 
programs to detect unrestricted and unsupervised manufacture and sale of drugs that may 
contain inorganic arsenic must be implemented to prevent a potentially fatal disorder. 

1 1 . Acknowledgment 

Arsenic is a fascinating element. We were inspired by treating patients having contact to 
arsenic. Quod vide: 

Shab, C. Crofimann und C. Bayerl, „ Multiple Basalzellkarzinome und aktinische Keratosen 
bei einem landwirtschaftlichen Arbeiter nach Arsen-Exposition: Immer nur BK 1108?" 
Dermatologie in Beruf und Umwelt (Vol. 57,No. 4/2009(4. Quartal)). 

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arsenicism: review of seventeen cases," J.Am.Acad.Dermatol, 38(2 Pt 1), 179-85. 
Wong, S. S., K. C. Tan, and C. L. Goh (1998c), "Cutaneous manifestations of chronic 

arsenicism: review of seventeen cases," J.Am.Acad.Dermatol, 38(2 Pt 1), 179-85. 
Yang, H. T., H. J. Chou, B. C. Han, and S. Y. Huang (2007), "Lifelong inorganic arsenic 

compounds consumption affected blood pressure in rats," Food Chem.Toxicol, 

45(12), 2479-87. 
Yoshida, T., H. Yamauchi, and Sun G. Fan (2004), "Chronic health effects in people exposed 

to arsenic via the drinking water: dose-response relationships in review," 

Toxicol. Appl.Pharmacol, 198(3), 243-52. 
Yousef, M. I., F. M. El-Demerdash, and F. M. Radwan (2008), "Sodium arsenite induced 

biochemical perturbations in rats: ameliorating effect of curcumin," Food 

Chem. Toxicol, 46(11), 3506-11 . 



11 



Freshwater Decapods and Pesticides: 
An Unavoidable Relation in the Modern World 

Leandro Negro 1 , Eloisa Senkman 13 , 
Marcela Montagna 1 and Pablo Collins 1 ' 2 ' 3 

1 Instituto National de Limnologia, Ciudad Universitaria Pje El Pozo, 

2 Facultad de bioquimica y Ciencias Biologicas Universidad National del Litoral, 

3 Facultad de Ciencias y Tecnologia Universidad Autonoma de Entre Rios, 

Argentina 



1. Introduction 

1.1 Levels of organisation in biological systems and their relationships 

Scales in nature can be difficult to define and understand because several ecological factors 
can interact. The study of different biological scales contributes to information that varies in 
its quality and significance for humans. Observations at the ecosystem scale are of great 
ecological significance but can be of low quality or provide little information about causes; 
at the other extreme, molecular studies that provide exact determinations of causes can have 
very little relevance to effects at a larger scale (Figure 1). In the middle of these extremes are 
observations that provide more or less significant and relevant information. Increasing the 
level of biological complexity in our observations can lead to an unexpected increase in the 
number of variables to be considered, requiring the consideration of n-adimensional 
conditions. 



O 


significance 
ecological 


JKecosystemic 
' level 


01 


molecular ^B^^ 
level ?J0^^ 
cause 
knowledge 








spatial scale 



Fig. 1. Relationship between spatial and temporal scales with the quality information 
obtained 

Each level of study is influenced by the level below it, and each level affects the level above 
it, which is mediated by interspecific relationships that influence ecosystem structure, and 
this can vary according to the heterogeneity of an ecosystem. When biological complexity 



198 Pesticides in the Modern World - Risks and Benefits 

increases, it is important to consider that temporal and spatial dimensions are 
interconnected, e.g., molecular reactions occur in spaces smaller than one-hundredth of a 
millimetre and at reaction times of less than one second. At the same time, the effects of the 
predation of one species on the populations of other species may play out in spaces at the 
scale of kilometres and at timescales that can exceed a year. According to the heterogeneity 
of a system, its component species and the processes involved, variations in the time and 
space involved in a given process can be very important (Figure 1). 

1.2 Fauna in aquatic systems 

Year after year, the quality of aquatic environments is recognised as a priority for humanity, 
with particular emphasis on the quantity and quality of freshwater. 

Among the faunal components of aquatic environments, decapods, an order of crustaceans, 
are an interesting group that possesses biological characteristics useful in assessing the 
quality of inland aquatic systems. In addition, some species of decapods may be used as 
food by humans and are part of the food chain of other species used by humans as food, 
mainly fish and birds. 

Five decapod families occur in southern South America and east of the Andes. Some of these 
decapods are endemic at the family level, others at the genus level and still others at the 
species level. These families include prawns and shrimp (Palaemonidae and Sergestidae), 
crabs (Trichodactylidae), pseudocrabs (Aeglidae) and crayfish (Parastacidae) (Collins et al., 
2007). 

Some of these families live in burrows constructed of fine sediment (some Trichodactylidae 
and Parastacidae). Others live in the background using clasts, rocks or tree trunks for hide 
under this cover (Aeglidae). Some decapod families live among aquatic vegetation (some 
Trichodactylidae and Palaemonidae), while others live all or part of their lives in the water 
column (some Palaemonidae and Sergestidae). Thus, the habitats used by this group are 
very diverse, and different taxa have different relationships to the land environment. The 
densities of decapods can be very high at certain times of year and may exceed 500 animals 
per square meter (e.g., Palaemonidae). Their diets are varied and may include plant matter 
(e.g., aquatic plants and phytoplankton debris), microinvertebrates (e.g., protozoa, 
cladocerans, rotifers, and copepods), macroinvertebrates (Palaemonidae insect larvae, 
oligochaetes, molluscs) and vertebrates (fish). The trophic resource used by decapods is 
mainly composed of live animals, but dead animals are also commonly fed upon. 
Consumption intensities are very high, transferring energy and material from various 
bottom levels (e.g., oligochaetes, chironomid larvae, zooplankton, and vegetal remains) to 
the top trophic levels (e.g., fish, mammals, reptiles, birds) (Collins & Paggi, 1998; Collins 
1999; Williner & Collins, 2002; Collins 2005; Collins et al., 2006). 

Since the industrial revolution, the human population has been growing rapidly and has 
therefore required more intensive management of natural environments. This need for 
intensive management has included the use of more land for growing food, causing the 
conversion of forests, jungles, and grasslands, among other ecosystems, into farmland. 
Subsequently, different poisons (e.g., herbicides, insecticides, fungicides) have been 
employed with the aim of eliminating those plants and animals that could use the crop 
resources (cereal or other crops), which humans call "pests". The use of these chemicals grew 
during the last century in an unprecedented manner in both volume of use and in the 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 



199 



different formulations available. In addition, the creation of new compounds, the genetic 
modification of plants to withstand pesticides, and the improved effectiveness of the 
methods of pesticide application have had substantial economic support for research and 
development. Unfortunately, studies of the damage caused by these chemicals have not had 
similar financial support. 




Fig. 2. Land use in South America indicating the surface of Orinoco, Amazon and La Plata 
basins, approximately. A) urban development and level (A) farm and industrial activities 
(B). Scale indicates differences in the intensities of the activities (modifies of Collins et al, in 
press). 

Thus, the man invades and uses natural systems, intensively modifying them, and the fauna 
suffers extreme stress. Aquatic systems, although generally not targeted by direct 
application of pesticides, are impacted as a result of runoff after rain. This water, with all the 
elements that may be associated with it, goes into rivers or depressed areas. Groundwater 
may also be contaminated by percolation after rainfall. This mobility of elements occurs 
more intensively when the fields have no vegetative cover. 

Moreover, the higher populations in cities have caused an increase in the urban area 
required to accommodate people and their families. This, together with increased biocide 
use and the increased area of impermeable surfaces in cities has meant that household 
chemicals and waste products are transported rapidly to aquatic systems during rains (Jose 
de Paggi et al., 2008). 

Watersheds are continually being impacted, and care must be taken to ensure their quality 
control because these watersheds provide people with water to live. In South America, there 
are three major basins with water flows ranging from 18.000 nr 3 s -1 to approximately 220.000 
m- 3 s-! (Bonetto & Waiss, 1995; Lewis et al., 1995). These basins are the Amazon, Orinoco and 
La Plata. Of these three, the most densely populated watershed, with the greatest number of 
agricultural enterprises and the largest number of factories, is La Plata Basin (Figure 2) 
(Collins et al., in press). 



200 Pesticides in the Modern World - Risks and Benefits 

1.3 Biocides 

The variety of active ingredients used as biocides and their commercial formulations, 
solvents and coadjuvants or related chemicals is immense. All of them are used by 
application with agricultural aircraft, sprayers, hand-held units, or trucks that carry the 
spraying equipment, according to the extension land, application protocols, crop types and 
soil characteristics. Studies on native fauna are scarce, and only for very few taxa have the 
biological effects of biocides been studied. Studies on the interrelationships among the fauna 
components in relation to pesticide use have also been scarce. The actions of each biocide 
cause different biological responses, e.g., cypermethrin provokes an increase in metabolic 
activity and glyphosate a decrease (Collins et al., in press). The action of each pesticide is 
different, and the scarce information in their effects makes it very difficult to recognise the 
magnitude of the harm caused by these biocides on non-target species and on aquatic 
environments. The studies that have been conducted have focused on assays involving the 
active ingredient; however, it is not only the active ingredients that cause damage to the 
environment but also those compounds that are in the formulation and are considered inert. 
These compounds can increase the toxicity of the active ingredient, facilitating its ingression 
in biological systems, or may be toxic by themselves. It is therefore necessary for studies not 
to ignore commercial formulations, because they may include several compounds that can 
affect aquatic systems. 



Biocides 

(active ingredient and others elements ???) 



Ht 



Abiotic factors 

* temperature 

* water level 
Might 

* sediment types 



If /r '^^f^V) 



(I 




Biotic factors 
+ predation 
+ refuge 

+ trophic resource 
+ reproduction 

Fig. 3. Structure of typical area more affect by biocides through of sprayer with airplane, 
runoff after rain or groundwater potentially contaminated 

2. Biocides in organisms: internal actions 

2.1 Uptake at the tissue and cellular level 

The toxicants direct absorption from the water by the integument and gills of crustaceans 
and/ or through the ingestion of contaminated food via the gastrointestinal tract can cause 
serious toxicity to normal biological functions at the tissue, cellular, and molecular levels. 
However, the permeability of biological barriers and the rate of transport of chemicals into 
an organism are affected by the metabolic activity of the animal and, indirectly, by factors 
influencing this activity (water temperature, pH, hardness, the presence of other chemicals). 
The metabolic activity of the animal is influenced by its body size, growth rate, physical 
activity, and physiological state (juvenile or mature, moulting, feeding) (Zitko, 1980). 
As the mechanisms of the toxic action of many pesticides usually occur on the surface of or 
inside the cells (Fent, 2004), the movement of these xenobiotics across membranes depends 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 201 

on the chemical nature of the pesticide involved. Matsumura (1977) summarised the specific 
properties that influence uptake into aquatic organisms: lipid and water solubility, chemical 
stability against degradative action by biological systems (biotransformation), and the 
molecular weight of the chemical. These physicochemical properties determine the affinities 
of toxic compounds for the materials comprising the arthropod cuticle and plasma 
membrane of the cell (Hartley & Graham-Bryce, 1980). 

Because lipids constitute a substantial part of the plasma membrane, lipid solubility is a 
very significant factor determining the rate of penetration of many toxic compounds (such 
as organochlorine pesticides) by passive diffusion through the non-polar portion of the 
membranes. Lipid solubility is usually characterised by the octanol/water partition 
coefficient (K ow ). In other cases, both facilitated diffusion and active transport are required 
for the passage of toxic into the cell through channel proteins and via their association with 
carrier proteins, respectively (Newman & Unger, 2003). The passage through a protein 
channel occurs down a concentration gradient that may be subject to saturation kinetics, and 
it is influenced by the size of the molecule, which determines a lower permeability of the 
membrane with increasing molecular size (Zitko, 1980). Moreover, the uptake of several 
pesticide compounds requires an active process with an expenditure of metabolic energy in 
living tissue. Through these pathways, toxicants enter cells and cause alterations in the 
physicochemical properties of the cytoplasm and the pH of the medium, destruction of the 
membranes of the organelles, disruption of the normal functioning of the cell proteins, and 
inhibition of the actions of the enzymes (Sohna et al., 2004; Collins, 2010). 
Because in multicellular organisms the distribution of toxicants occurs in more than one 
compartment, within the crustacean body, haemolymph circulation may be involved in the 
transport of these chemicals to their sites of action and even more so if it is an open system 
that flows around the organs. In other arthropods, such as insects, Brooks (1974) reported 
that phosphoric acid penetrates the cuticle more rapidly than organochlorine insecticides, 
and having passed this barrier, the toxicant enters the haemolymph and may be transported 
to all parts of the organism in solution, if water soluble, or bound to proteins or dissolved in 
lipid particles, if lipophilic. The relatively hydrophilic molecules are much more likely to 
remain in this circulatory fluid than small, hydrophobic molecules, which are rapidly 
distributed in several organs and stored in lipid tissue (Hartley & Graham-Bryce, 1980). 

2.2 Toxicity and biotransformation 

The adverse effects of toxic products on crustaceans depend on its concentration and 
affinity, activity (intrinsic toxicity, which is function of molecular structure) and chemical 
biotransformations (James, 1987) and the acclimation responses of the individual (Klerks, 
1999). For biocides, such as organophosphates and carbamate anticholinesterases (anti- 
ChEs), intrinsic toxicity can be judged by measuring the inhibition of cholinesterase and 
propagation of action potentials on synaptic transmission (see biomarkers section). 
While some organic compounds are sufficiently water-soluble (hydrophilic) for excretion 
and can be eliminated rapidly, many lipophilic components cannot be directly excreted and 
would accumulate if not processed to more polar derivatives. Because the unaltered toxicant 
and any of its transformation products (metabolites) may be excreted, excretion represents a 
possible protective mechanism against the toxicant (Newman & Unger, 2003). Usually, 
organic pesticides are subject to modifications through enzyme-catalysed 
biotransformations leading to detoxification or activation (Figure 4). Chemical 



202 Pesticides in the Modern World - Risks and Benefits 

biotransformation in animals occurs via Phase I (functionalisation) and Phase II 
(conjugation) reactions, which are more readily excreted than the parent compound (Brooks, 
1974; Oesch & Arand, 1999). 

1. Phase I reactions. In this phase, several enzymes introduce a polar reactive group to the 
molecule, making it more water soluble while also increasing the possibility of further 
metabolism by Phase II enzymes. Two major groups of enzymes involved in Phase I 
metabolism include oxidoreductases and hydrolases that are located in the endoplasmic 
reticulum of the cell in many organs and tissues (James, 1987). 

1. The oxidoreductases include the quantitatively most important superfamily of 
xenobiotic-metabolising enzymes, the cytochrome P450-dependent 
monooxygenases (CYP), flavin-containing monooxygenases (FMO), monoamine 
oxidases (MAO), and cyclooxygenases (COX), all of which introduce oxygen into or 
remove electrons from their substrates, with a few exceptions. 

2. The dehydrogenases and reductases, such as alcohol dehydrogenases, aldehyde 
dehydrogenases, and carbonyl reductases, add or remove hydrogen atoms to or 
from the target molecule. The hydrolases comprise families of enzymes specialised 
in the hydrolysis of esters, amides, epoxides, or glucuronides (Oesch & Arand, 
2005). 

The predominant functions of Phase I reactions are the conversion of polar, lipophilic 
compounds into more polar, more hydrophilic compounds and the introduction or 
liberation of functional groups that can be used for conjugation in the subsequent Phase II of 
xenobiotic metabolism. 

2. Phase II reactions. Phase II enzymes often conjugate the polar groups produced by 
Phase I enzymes to introduce more bulky hydrophilic substituents, such as sugars, 
sulphates, or amino acids, into the molecule. This conjugation substantially increases 
the water solubility of a chemical, making it more easily excreted. The conjugation of 
the xenobiotic metabolism is carried out by transferases. 

1. Electrophilic substrates are taken over by the glutathione S-transf erases (GSH S- 
transf erase). 

2. Nucleophilic substrates (i.e., those with hydroxyl, sulfhydryl, amino, or carboxyl 
groups) are metabolised by UDP-glucuronosyltransferases (UGT), sulfotransferases 
(SULT), acetyltransferases (AT), acyl-CoA amino acid N-acyltransferases, and 
methy ltransf erases . 

Phase II involves reactions such as glycosylation, sulfation, mercapturic acid formation, 
amino acid conjugation, and acetylation. Carboxylic acid groups in xenobiotics can be 
conjugated with amino acids prior to excretion (Tang et al., 2005). Metabolites formed by 
conjugation reactions are usually less toxic than the unconjugated compound, although 
there are notable exceptions to this rule (James, 1987). In addition, the metabolic events that 
increase the water solubility of a chemical usually cause a significant reduction in its 
biological half-life by making it more readily excreted (Brooks, 1974). 

However, the patterns of activity of key enzymes involved in the detoxification of pesticides 
can be modified by the same toxic effect of xenobiotics. An elevation in glutathione S- 
transferase (GSH S-transferase) levels in the hepatopancreas and gills was reported for 
freshwater prawns (Macrobrachium malcolmsonii) and crabs (Paratelphusa hydrodromus) 
exposed to endosulfan, reflecting the formation of glutathione (GSH) and endosulfan 
complexes as a means of detoxification/ elimination (Yadwad, 1989; Saravana Bhavan & 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 



203 



Geraldine, 2001). Conjugation of xenobiotics with reduced glutathione (GSH), catalysed by 
glutathione S-transferase (GSH S-transferase), is an important physiological process in the 
elimination of toxic substances from the body. These authors suggest that the activation of 
such a mechanism probably confers cytoprotection against endosulfan-induced cellular 
stress. 

There are some toxicants in which biotransformation through either Phase I or Phase II can 
produce a highly reactive chemical, for example, the organophosphorus compounds. 
Although many of the insecticides in other chemical classes are toxic in their original parent 
forms, this is not true for many of the organophosphorus insecticides, especially those of the 
phosphorothioate configuration (such as parathion, chlorpyrifos, and diazinon), 
characterised by a P=S group. The insecticides possessing a P— S group are usually not very 
potent anti-ChEs, and they require bioactivation of their P=0 metabolites, called oxons, to 
display appreciable anti-ChE potency (Tang et al., 2005). This bioactivation (reaction of 
desulfuration) is mediated by cytochrome P450-dependent monooxygenases through an 
attack on the sulphur by oxygen to create an unstable phosphooxythiiran intermediate (a 
three-membered ring composed of P, O, and S) that subsequently decomposes to the oxon 
(P — O) metabolite plus an active form of S (S:). In addition, the S is reactive in the tissues 
and is capable of damaging some proteins, including the cytochrome P450-dependent 
monooxygenases. 



ABSORPTION 





ORGANIC COMPOUND 



PHASE II 
Siucosylation 
ACTIVATION H -| (9l"turan^^|lu C Mi*ltan) 

Mercapturic Acid Biosynthesis 

Acelylalion 

Amino Acid Conjugation 



ELIMINATION 



POLLUTED ENVIRONMENT 




Fig. 4. Different process that can occur in decapods when the animals are affected by some 
biocide. 

Other modifications to the toxic action of xenobiotics in crustaceans may occur via the 
phenomenon of physiological acclimation. In this case, an individual organism that becomes 
exposed to a specific contaminant may be less severely affected by this contaminant if it had 
been previously exposed to it. This effect is generally the result of the induction of a 
detoxification mechanism, as cytochrome P450, in response to the initial exposure (Tang & 
Garside, 1987; Stuhlbacher et al., 1992). Klerks (1999) observed (in the shrimp, Palaemonetes 
pugio) that acclimation results in an increased resistance at only a limited range of 
concentrations, with generally no change in resistance at lower pre-exposure levels and a 



204 Pesticides in the Modern World - Risks and Benefits 

decreased resistance at higher pre-exposure concentrations that are stressful or result in a 
significant increase in contaminant body burdens. Such resistance occurs for some 
contaminants but not for others, and a lack of acclimation to complex mixtures occurs 
because positive responses to one contaminant are offset by negative responses to another 
contaminant. According to this observation, this can be explained by the fact that the 
energetic costs resulting from exposure to one contaminant (either for damage-repair 
functions or for detoxification processes, such as the production of P450 oxygenases) would 
compete with the energetic requirements associated with exposure to the other contaminant. 

2.3 Biomarkers 

To evaluate effects of pollutants on animal populations, communities and ecosystems, 
various methods have been developed, ranging from the (sub)cellular to the ecosystem level 
of biological responses. However, the predictive ability of measurements at higher levels of 
biological organisation is limited because ecologically important effects (e.g., death or 
impaired organismal function) have already occurred before they can be detected at 
population and community levels. In recent decades, biomarkers at suborganismal levels of 
organisation (biochemical components or processes, physiological functions, and 
histological structures) have been considered to be viable measures of responses to stressors 
(Hansen, 2003). These indicators of stress responses are useful in assessing the short-term 
well-being or long-term health status of an animal (Paterson & Spanoghe, 1997). 
Metabolic changes observed in crustaceans exposed to pesticide pollution create widespread 
disturbances in general physiological processes, such as enzymatic activities, oxygen 
consumption, and changing energetic requirements. Some of the standardised types of 
biomarkers are those linked to disturbance to osmoregulation and water balance/ ion- 
homeostasis, cholinesterase inhibition activity, protein stress, oxidative stress, and endocrine 
disruption. 

2.3.1 Haematological parameters 

Alterations in the haemolymph protein, haemocyanin, osmolality, ion compositions, total 
haemocyte counts, differential haemocyte counts, total free amino acid, nucleic acids 
(concentrations of DNA and RNA), phenoloxidase (PO) activity, and superoxide anion (O2") 
may occur in crustaceans as a result of toxicant expositions. Yeh et al. (2005) reported a 
significant depression in haemolymph osmolality that mainly resulted from a decrease in 
the haemolymph chloride concentration (CH) in the prawn, Macrobrachium rosenbergii, after 
8 days of exposure to sublethal concentrations of trichlorfon. However, a decrease in 
haemolymph pC>2 was found among these prawns, which may be related to decreased 
ventilation and impeded respiratory gas exchange, leading to respiratory disturbances via 
the inhibition of respiratory mechanisms and damage to respiratory organ epithelial cells. 
Similarity, a decrease in the pH and HCO3- of the haemolymph induced an increase in the 
pCC>2 level, benefiting the excretion of CO2 in the haemolymph and resulting in a decrease 
in TCO2, suggesting that trichlorfon disturbs the extracellular acid-base balance of prawns. 
In crustaceans, gill lamellae and epipodites are involved in osmoregulation, and the 
histopathological changes in these structures (haemocytic congestion, gill lamellae necrosis, 
and the accumulation of particles surrounding the gill lamellae) were observed with lethal 
concentrations of fenitrothion (Lignot et al., 1997). According to these authors, the presence 
of particles surrounding the gill lamellae may have been a consequence of a lack of 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 205 

ventilation in the branchial cavity due to the inhibitory action of the pesticide on the 
nervous system. In contrast, Saravana Bhavan & Geraldine (2001) observed in the prawn, M. 
malcolmsonii, an increase in the content of total free amino acid in the haemolymph as a 
result of protein degradation. In addition, the accumulation of soluble protein suggests that 
this was necessary to serve as a compensatory pool to restore enzymes lost to tissue necrosis 
and to provide prawns with the energy required to cope with the stress of exposure to 
endosulfan. 

2.3.2 Cholinesterase (ChE) activity (inhibition) 

Cholinesterases are serine hydrolase enzymes and degrade the neurotransmitters in 
cholinergic synapses. The toxicity of some pesticides, such as organophosphates and 
carbamate insecticides, is mainly caused by the inhibition of ChE activity of vertebrates and 
invertebrates. This inhibition leads to the accumulation of acetylcholine in the synaptic 
terminals and therefore to a change in the normal transmission of the nervous impulse. This 
interference may result in neurological manifestations, such as irritability, restlessness, 
muscular twitching, and convulsions, that may end in the respiratory failure and death of 
the animal (WHO, 1986). Consequently, most studies describe the use of ChE levels as a 
biomarker of exposure and/ or the effect of several pesticide compounds in aquatic species. 
However, distinct enzyme isoforms with different sensitivities towards anticholinergic 
contaminants may exist, depending on the species. These isoforms are usually divided into 
two broad classes: acetylcholinesterases (AChE) and bufyrylcholinesterases (BChE), which 
are distinguished primarily based on substrate specificity (Sulfa tos, 2005). 
In crustaceans, published studies have also shown mixed results with regard to substrate 
preference. Fulton & Key (2001) reported that AChE in Palaemonetes pugio hydrolyses 
acetylcholine iodide (ACTH) and acetyl-b-methylthiocholine iodide (AMTH) much faster 
than other choline esters (such as propionylcholine) and is inactive on butyrylcholine. In 
contrast, BChE not only hydrolyses butyrylcholine but may also hydrolyse acetylcholine. 
The two enzyme isoforms may also be distinguished by their susceptibility to selective 
inhibitors; l,5-bis-(4-allydimethyl-aminoniumphenyl)-pentan-3-one dibromide (BW284c51) 
and tetraisopropyl pyrophosphoramide (/so-OMPA) are selective inhibitors for AChE and 
BChE, respectively (Sultatos, 2005). 

Organophosphates are generally irreversible inhibitors because the dephosphorylation rate 
of the bound enzyme proceeds at an insignificant rate. Therefore, the inhibitory effects of 
organophosphate exposure may be long lasting, with recovery depending on new enzyme 
synthesis (Habig & Di Giulio, 1991). Several studies with prawn, crab, and lobster species 
have shown that AChE inhibition in the animals still occurred days after exposure had 
ended (Reddy & Rao, 1988; McHenery et al., 1991; Abdullah et al, 1994; Key & Fulton, 2002). 
A slow time course for recovery of depressed AChE levels may cause exposed organisms to 
be susceptible to other anthropogenic or natural hazards or to exhibit behaviours not 
conducive to maintaining the population. 

2.3.3 Stress proteins 

The most abundant and widely studied group of stress proteins is the hsp70 (heat shock 
protein 70) protein family. The cellular functions of these proteins include the stabilisation 
of unfolded protein precursors before assembly, translocation of proteins into organelles, 
rearrangement of protein oligomers, dissolution of protein aggregates, and refolding or 



206 Pesticides in the Modern World - Risks and Benefits 

degradation of denatured proteins (Feige & Polla, 1995). Induction of stress protein 
synthesis by pesticides is reported to be highly tissue-specific in aquatic animals. Among the 
tissues analysed (gill, skeletal muscle and hepatopancreas) by Selvakumar et al. (2005) in 
Macrobrachium malcolmsonii, induction of hsp70 synthesis was recorded only in the gill tissue 
of prawns that had been exposed to sublethal concentrations of endosulfan. In contrast, 
exposure of prawns to sublethal concentrations of carbaryl failed to elicit hsp70 synthesis in 
any of the three tissues analysed. 

2.3.4 Oxidative stress 

Under normal conditions, equilibrium exists between the amounts of free radicals generated 
and antioxidants available to quench or scavenge them, thereby protecting the organism 
against the deleterious effects of pollutants. However, oxidative stress occurs when the 
critical balance between oxidants and antioxidants is disrupted as a result of the depletion of 
antioxidants or excessive accumulation of the reactive oxygen species (ROS), or both, 
leading to damage to macro molecular components (Scandalios, 2005). Many xenobiotics, 
such as pesticides, may cause oxidative stress, leading to the generation of ROS and 
alterations in antioxidants or free oxygen radicals scavenging enzyme systems in aquatic 
animals (Dettbarn et al., 2005). However, the cells of crustaceans possess a variety of 
chemical and enzymatic mechanisms to protect them from oxidative damage. These 
mechanisms include an enzymatic antioxidant defence system comprising enzymes such as 
superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione S- 
transferase (GSH S-transferase) and non-enzymatic antioxidants like glutathione (GSH), 
ascorbic acid (vitamin C) and a-tocopherol (vitamin E), which are capable of neutralising or 
scavenging the reactive oxygen species (Vijayavel & Balasubramanian, 2009). These authors 
showed that the toxicity of fenvalerate to the prawn, Penaeus monodon, led to a significant 
induction of lipid peroxidation and GSH S-transferase activity in the hepatopancreas, 
muscle and gills. On the contrary, the activities of SOD, CAT, glutathione peroxidase, 
vitamin C, vitamin E and GSH were reduced in prawns exposed to sublethal concentrations 
of fenvalerate. 

2.3.5 Neuroendocrine systems 

Toxicity induced by a pesticide is the result of interaction of the compound or one of its 
metabolites with the biochemical events involved in the homeostatic control of a 
physiological process (Newman & Unger, 2003). Physiological processes are mostly 
coordinated by hormones. Therefore, the effects of organic compounds on functions 
regulated by hormones in crustaceans could be used as biomarkers of environmental 
pollutants. 

According to Rodriguez et al. (2007), endocrine disruption can take place at different 
physiological levels: 1) altering (inhibiting or stimulating) the secretion of hormones; this 
possible effect is related to mechanisms that control both the release of hormones from 
endocrine cells and the synthesis of these hormones; 2) interfering with hormone-receptor 
interaction; in this sense, endocrine-disrupting compounds (EDCs) can act as agonists or 
antagonists by directly binding to a hormone receptor. Indirectly, however, an EDC could 
interfere via several mechanisms at any step of the transductional pathway of a hormone, 
therefore altering its final effect; 3) modifying the metabolism of circulating hormones, that 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 207 

is, by increasing or decreasing their excretion rates and/or biotransformation in the liver, 
hepatopancreas or other organs. 

Neurosecretory structures (X-organ-sinus gland) in the eyestalk are the most important 
components of the neuroendocrine system of the stalk-eyed crustaceans. The main 
hormones secreted by the sinus gland are the following: MIH (moult-inhibiting hormone), 
GIH (gonad-inhibiting hormone), MOIH (mandibular-organ-inhibiting hormone), CHH 
(crustacean hyperglycaemic hormone), several colour change hormones (controlling 
pigment migration) and NDH (neurodepressing hormone). Some of these hormones have 
a second endocrine gland as their target (MIH, GIH, MOIH), while the others have 
somatic tissues as targets. MIH, GIH, MOIH and CHH belong to a single family of 
peptides (Fingerman et al., 1998; Chang, 2001). These neuropeptides, synthesised in the 
XO (X-organ), a cluster of neuron perikarya located in the medulla terminalis of the 
eyestalk, are transported to and stored in the axon terminals, forming a neurohaemal 
organ named SG (sinus gland) and released by exocytosis into the haemolymph 
(Lorenzon, 2005). 

The CHH have been shown to regulate carbohydrate metabolism in the shore crab, Carcinus 
maenas; the kumuran prawn, Penaeus japonicus; the lobster, Homarus americanus; the 
freshwater crab, Oziotelphusa senex senex; and the fiddler crab, ilea triangularis (Kegel et al., 
1989; Lorenzon 2005; Purna Chandra Nagaraju et al., 2005). The neurotransmitter, 5-HT 
(serotonin), plays a fundamental role in hormone (CHH) modulation, and at the same time, 
pollutants can alter their level and function. Therefore, 5-HT has been known to have a 
potent hyperglycaemic effect with increases in the glucose haemolymphatic concentration 
resulting mainly from the stimulation of glycogen breakdown in the hepatopancreas 
(Fingerman et al., 1998). Hyperglycaemia is a typical response of several crustacean species 
to chemical stressors, including some pesticides, hydrocarbons and heavy metals. However, 
several reports have shown that an increased haemolymphatic level of glucose alone does 
not necessarily prove that there was a disruptive effect on the endocrine system. Because 
CHH is released to raise glycaemia as an adaptive response to several stimuli (such as 
emersion, starvation, critical temperatures and others), this hormone has been proposed as 
functioning as a crustacean stress hormone (Chang, 2001). 

2.4 Histological effects 

Crustaceans are considered as carrying a simple and primitive immune system (Fig. 5). The 
hepatopancreas is known as the detoxification site and also as a sensitive organ to stress, as 
it quickly responds to exposure to noxious compounds. 

The hepatopancreas is essentially composed of branched tubules and of 4 types of epithelial 
cells: embryonic cells (E-cells), fibrillenzellen cells (F-cells), restzellen cells (R-cells) and 
blasenzellen cells (B-cells). E-cells are the only ones showing mitotic activity, being 
important in dead cell replacement. R-cells have absorptive functions supported by the 
presence of lipid droplets in the cytoplasm. These cells are involved in the delivery of 
nutrients to other organs via the haemolymph; the nutrient reserves are mobilised through 
R-cells to provide energy to the rest of the body. In addition, R-cells are interpreted as sites 
of intracellular waste deposition characterised by autophagosomes and residual bodies. 
These cells detoxify heavy metals and other lipophilic compounds by their accumulation in 
a soluble form in the cytoplasm, followed by excretion. F-cells are where protein synthesis 
and enzyme production occurs (Sousa et al, 2005). 



208 Pesticides in the Modern World - Risks and Benefits 

Exposure to pesticides causes an imbalance in epithelial cells. Among the effects found, 
biocides cause an increase in R- and F-cells and an inhibition in E-cells. An R-cell increase in 
response to noxious compounds may be related to two different strategies. More R-cells may 
increase the detoxification rate because a higher number of cells increases detoxification. 
However, noxious compounds cause effects not only in the hepatopancreas but also in gills, 
gonads, and other organs. As other body parts require energy to recover from deleterious 
effects, the R-cell number increases for transporting energetic resources, i.e., lipids. When 
submitted to pesticides, F-cells increase for the production of more enzymes as a way of 
deactivating toxic compounds. R- and F-cells increase because both cellular types play roles 
in detoxification, and each one develops a different action for the same purpose. A decrease 
in E-cells becomes important if we consider that pesticides cause necrosis and increase 
cellular apoptosis. These cells replace dead cells with new ones, trying to mitigate cell loss. 
Exposure to pesticides also causes haemocytic infiltration in the interstitial sinus, abnormal 
lumen of the tubules, separation of necrotic cells from basal laminae, thickened basal 
laminae, necrotic tubules containing tissue debris, melanisation and coagulation in the 
thickened basal laminae and walling off of the tubules by haemocytes around the thickened 
basal laminae. All these effects combined may cause deficiencies in hepatopancreas 
function, with in turn may cause death (Saravana Bhavan & Geraldine, 2000, Bianchini & 
Monserrat 2007, Collins 2010). 

In gills, one of the most important intake sites, biocide exposure also causes several 
histological damages, which in turn may cause functional deficiencies. Haemocytic 
infiltration in the haemocoelic space, swelling of the gill lamellae, lifting of lamellar 
epithelium, fusion of lamellae, abnormalities in the histoarchitecture, necrosis and other 
malformations are some the effects produced by pesticides in freshwater prawns and crabs. 
Gills are related to the transport of respiratory gases, their obvious function, and also with 
ammonia excretion, as the majority of waste nitrogenous compound excretion occurs 
through the gill epithelium. Gill damage may also cause difficulties in oxygen intake, 
eventually asphyxia, and disrupt osmoregulatory function. A decrease in oxygen 
consumption may cause progressive internal hypoxia, with several effects such as 
metabolism shifts and locomotive difficulties. Crustaceans generally maintain an aerobic 
metabolism as a way of obtaining energy from food reserves. Aerobic metabolism, through 
the Krebs cycle, provides more energy than anaerobic metabolism. However, this kind of 
cellular "respiration" requires enough oxygen to be developed. When the amount of oxygen 
needed for the maintenance of aerobic metabolism in not achieved, crustaceans obtain 
energy by glycolysis, an anaerobic metabolism of carbohydrates. This type of metabolism, 
although it allows individuals to obtain energy for vital actions, has two serious effects: 
lactic acid release and an underutilisation of the energy accumulated. While in aerobic 
metabolism, animals obtain 36 mol of ATP from 1 mol of glucose, in anaerobic metabolism, 
they obtain only 2 mol of ATP from 1 mol of glucose, with the production of 2 mol of lactic 
acid (Schmidt-Nielsen, 1997). In animals with sporadic hypoxia, lactic acid is used as a 
substrate for further oxidation, completing the Krebs cycle and gaining the full energy value 
of the original carbohydrate substrate. However, in animals with oxygen intake decreased 
by histological damage, hypoxia may be not temporary; if they continue to be exposed to the 
aggressor agent, gills are not able to reconstitute itselves, or recuperation time is not quick 
enough to supply the oxygen demand. The continuous internal hypoxia may provoke a 
constant release of lactic acid as metabolic waste, with the consequent acid imbalance. This 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 



209 



imbalance and its histological effects may eventually cause the death of the affected 
individual because of acidosis or progressive asphyxia (Vonk, 1960, Schmidt Nielsen, 1997). 



Effects in gills 

Hemocytic infiltration in 

the hemocoelic space 

Swelling of the gill 

lamellae 

Lifting of lamellar 

epithelium 

Fusion of lamellae 

Abnormalities in the 

histoarchitecture 

Necrosis 

Alterations in oxygen 

intake, ammonia excretion 

and ionic exchange 



Pesticides 



Effects In hepatopancreas 

Imbalance in cell type proportion 

Hemocytic infiltration in the interstitial 

sinuses 

Thickening of the basal laminae and 

separation of necrotic cells 

Formation of necrotic tubules 

Abnormal lumen 

Melanization and coagulation in the 

basal laminae 



Walling off of the tubules by hemocytes 
^=!> Effects in gonads 



Atresic and abnormally 
shaped oocytes 
Differences in oocyte size 
Decrease of viable oocytes 



Fig. 5. Histological pesticide effects in crustacean organs and its results. Crab image 
modified from Collins et al. (2004). 

Another effect of hypoxia is a decrease in locomotion. Crustaceans regulate their oxygen 
consumption within a range of dissolved oxygen concentrations. The minimal dissolved 
oxygen concentration within this range is called the critical oxygen concentration, below 
which crustaceans are not able to regulate their oxygen consumption. Given this situation of 
hypoxia, many crustaceans reduce their movements as a way of reducing the oxygen 
consumed by muscles, using the available oxygen instead of using it for metabolism (Zou et 
al., 1992, Zou & Stueben, 2006). The same response occurs if an animal has a deficiency in 
oxygen uptake, as in both situations the oxygen concentration in haemolymph is 
unsaturated. The oxygen deficiency causes a decrease in locomotive activities, with several 
effects. Animals are unable to escape from the contaminated area, exacerbating the effects of 
the contaminants. At the same time, a decrease in movement, as mentioned above makes 
animals more susceptible to predators. 

In addition to their respiratory function, gills play an important role in nitrogen compound 
excretion. Crustaceans are ammoniotelic animals, i.e., their nitrogenous metabolic end 
products are mainly excreted in the form of ammonia. The antennal gland plays the key role 
in body water and divalent cation regulation, but it plays a minor role in ammonia 
excretion, as in some cases less than 2% of the total ammonia is excreted in the urine via the 
antennal gland system (Parry, 1960; Cameron & Batterton, 2004). 

The high lipid solubility of ammonia makes it more diffusible through phospholipid 
bilayers. The mechanism supporting ammonia excretion in crustaceans is the simple 
diffusion of the non-ionic NH3 along a concentration gradient and the partial excretion of 
the ionised form NH4+, whose release through diffusion is facilitated because of its 
hydrophobicity (Weihrauch et al., 1999, 2004). 

Several aquatic crab species possess an excretion system based on the ionised form of 
ammonia, NH4 + , a water soluble compound which effluxes through the gill epithelium. 
Freshwater crabs have tighter gill epithelia than their marine relatives, developed to avoid 
ionic efflux and tolerate a hyposmotic environment. This epithelium is much less permeable 
by NH4 + , and freshwater crabs release their nitrogen compounds mainly as ammonia 
(Weihrauch etal., 1999). 



21 Pesticides in the Modern World - Risks and Benefits 

Concentrations of NH3 in the environment are kept low as a result of bacterial nitrification 
of ammonia to nitrite and nitrate, followed by the absorption of autotrophic organisms. This 
kind of environment favours ammonia excretion as a passive process driven by diffusion 
along a gradient. This process applies only to pelagic animals, generally prawns, colonising 
the water column, where the dilution and nitrification processes of aquatic biota keep 
ammonia concentrations really low. Benthic animals, such as crabs and crayfishes, are often 
faced with higher ambient concentrations of ammonia, present especially in anoxic, deep, 
stagnant water. Some species take refuge by hiding in riparian rocks and vegetation. Other 
species bury themselves in mud or, in the case of burrowing species, build extensive caves, 
in some cases more than 1 metre deep, with aerial or aquatic entry holes. They live in the 
bottom, where they find protection from predators and where the water is stagnant for 
hours. The very low water exchange rate, along with the fact that animals produce and 
excrete metabolic ammonia, increases the ammonia concentration and difficult simple 
diffusion. 

The process that these crustaceans uses to eliminate ammonia is active excretion. Ammonia 
excretion rates are correlated with Na+ absorption (Pressley et al., 1981, Harris et al., 2001). 
NH4+ substitutes for K+ in the activation of the ouabain-sensitive Na + /K + -ATPase, which is 
located in the basolateral membranes of the gill epithelium cells (Towle et al., 1981; Towle & 
Kays, 1986). This Na + /K + -ATPase is synergistically stimulated by NH4+ and K+. In 
freshwater decapods, at high NH4+ concentrations, the pump exposes a new binding site for 
NH4+ that modulates the activity of the Na + /K + -ATPase independently of K+ ions (Romano 
& Zeng, 2007, 2010). 

Histological damage in gills, and the mucus segregation observed in prawns exposed to 
pesticides, may hinder ammonia excretion. These effects are especially relevant in 
freshwater benthic crustaceans, mainly crabs and crayfishes. As mentioned above, the 
passive efflux of ammonium (NH4+) is difficult because of the thickened gill epithelium, 
while ammonia excretion (NH3) is difficult because of the environmental concentration. 
Nitrogenous waste compounds are eliminated by active efflux, and histological damage 
provoked by pesticides hinders this excretion process. When decapods are not able to 
eliminate the ammonia produced by nitrogen compound metabolism, it accumulates in the 
haemolymph, with several effects on individuals. Ammonia modifies the release of 
cytokines and increases the activity of lysosomal hydrolases. Ammonia toxicity is mediated 
by the excessive activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors in 
the brain. As a consequence, cerebral ATP is depleted, while intracellular Ca 2+ increases, 
with subsequent increases in intracellular K + and, finally, cell death (Weihrauch et al., 1999, 
2004). 

The intensity of the observed effects is related to pesticide concentration and animal 
resistance. Nevertheless, many of the described effects were achieved at concentrations that 
usually occur in the environment after aerial or terrestrial pesticide applications. The 
constant aggression provoked by biocides induces malfunctions in this vital organ, which 
eventually may cause the death of an individual. 

2.4.1 Histopathological effects on female gonads 

Freshwater decapods modified their reproductive strategy when they conquered freshwater 
environments. Larval stages were abbreviated or suppressed, and females invest their 
energy in fewer but more expensive progeny, which hatch at a more advanced stage. 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 21 1 

Gonads are characterised by fewer but bigger oocytes, with more energetic reserves for the 
extended embryonic stage. In subtropical regions, gonad development occurs during late 
winter, spring and summer, the same period when pesticide applications. The drift and 
runoff provoke the migration of biocides to aquatic environments, causing a continuous 
contact with females during gonad maturation. 

Ovary growth in crustaceans has two different periods: endogenous vitellogenesis 
(vitellogenesis I) and exogenous vitellogenesis (vitellogenesis II). The first period is 
characterised by an autosynthesis of lipovitellin and slow oocyte growth. The second period 
is characterised by the input of exogenous vitellogenin (a vitellin precursor) from outside of 
the ovary, mainly from the hepatopancreas, and rapid oocytic growth. Along with all the 
compounds provided by the hepatopancreas, lipophilic pesticides migrate to the ovaries 
(Lubzens et al., 1995). The effects of these biocides include abnormalities in shape, as the loss 
of the typical spherical shape of ovarian follicles; abnormal oocyte area increase or decrease, 
depending on pesticide type; and oocyte atresia (Rodriguez et al., 1994, Lee et al., 1996). The 
abnormal development of the ovaries causes a reduction in the available oocytes for 
fecundation, with the consequent reduction in eggs and the future brood, decreasing the 
population over the short and medium term. 

Once fecundation occurs, females carry their eggs in their pleon until juveniles or mysis 
hatch. If these females live in contaminated areas, the exposure to biocides causes 
different effects in eggs and embryos. The easiest observable effect is death, but embryo 
death may occur at relatively high pesticide concentrations. Eggs are surrounded by the 
chorion, which isolates them from the environment. In the case of freshwater decapods, 
the chorion is thicker than that present in marine decapods because it has to protect the 
embryo from the osmotic stress caused by the environment. This thicker chorion also 
isolates the embryos from biocides and other compounds (Lindley et al., 1999; Varo et al., 
2006). This protective effect makes embryos more resistant to toxicants, in some cases 
more resistant than juveniles, with a median lethal concentration similar to adults in 
several cases (Key et al., 2003; Li et al., 2006). Furthermore, embryos are more sensitive to 
pesticides when they are close to hatching because of the thinning of the chorion, which 
allows more pesticide to enter into the egg. This effect is also observable in prawns 
exposed to different salinity levels, as embryos are more sensitive to osmotic stress when 
they are close to hatching (Ituarte et al., 2005). 

In addition to lethality, constant exposure to pesticides may cause differences in incubation 
periods and several abnormalities in embryos. Among these abnormalities, biocides may 
cause hydropsy, abnormal eye spots and several atrophies in the eyes, the pleon and the 
dorsal spine (Rodriguez & Pisano, 1993; Lee & Oshima, 1998). All these abnormalities 
provoke the death of the juvenile, either from internal malformations of organs or from the 
incapability to moult successfully. Additionally, abnormalities in pleopods and pereiopods 
cause the inability to eat, find food or avoid predators. 

Constant exposure to pesticides causes a reduction in functional oocytes, resulting in fewer 
eggs, a reduction in surviving embryos and a decrease in juveniles that will reach the adult 
stage, which in turn provokes effects on populations, the community and the ecosystem. 

3. Reproduction effects 

Freshwater environments impose a severe osmotic stress to the animals living there. Marine 
crustacean reproduction is characterised by a large brood, which hatches as larvae and 



212 Pesticides in the Modern World - Risks and Benefits 

undergoes several stages up to the juvenile stage. Freshwater environments impose a severe 
osmotic stress on unprotected eggs and the free larvae stage. In the same way, developing 
embryos must be protected against this stress. When they conquered these environments, 
decapods developed different strategies to protect eggs and embryos. The primitive pelagic 
larval phases were suppressed; larval stages occur inside the egg, and the offspring hatch as 
mysis or juveniles. This internal development (i.e., inside the egg) imposes a greater 
protection to embryos against environmental pressures, especially in the susceptible larval 
stages. To support these internal stages, eggs increased in size and energy resources, mainly 
lipoproteins, because embryos grow inside the eggs and use their internal energy resources. 
Freshwater decapod females carry their eggs in the pleon, protecting them until the larvae 
or juveniles hatch. Because of their increased size, the number of eggs that a female can 
carry decreased, resulting in a concomitant decrease in the number of offspring (Ruppert & 
Barnes, 1994; Lee & Bell, 1999). 

Several pesticides are highly lipophilic and are accumulated mainly in lipid reserves. 
During ovary development, oocytes accumulate lipids and lipoproteins, mainly 
lipovitellin, forming the vellum, which in turn will be used by the embryo as an energetic 
resource (the embryo "feeds" on the vellum). Attached to the lipovitellins, pesticides enter 
to the oocytes and accumulate on them. One explanation for the relatively greater 
resistance of females to organic pollutants is the distribution of these toxicants in the 
ovary, decreasing their concentrations in vital organs such as the hepatopancreas and 
delaying death (Sheridan, 1975; Menone et al., 2000; Wirth et al., 2001; Menone et al., 2004, 
2006; Santos de Souza et al., 2008). The presence of pesticides in the oocytes implies that 
the embryo, beginning with fertilisation, is exposed to pesticides. Embryos grow and feed 
on the lipid reserves present on the vellum, with the consequent intake of pesticides. This 
may provoke not only the death of the embryos, with the release of dead eggs by the 
female, but also sublethal effects, such as abnormal size in eggs; deformation of embryos, 
such as tissue dropsy, atrophy, abnormal or depigmented eyes; and abnormalities in the 
pleon, telson, and spine, pereiopods and pleopods (Rodriguez et al., 1994; Saravana 
Bhavan & Geraldine, 2001). Deformation may cause difficulties in hatching or in brood 
survival, as activities such as swimming and searching for prey or escaping from 
predators may be hampered, and even moulting may not be successfully completed (Fig. 
6). 

In the case of freshwater decapods, the amount of vittelins and the time that embryos 
spend inside the eggs are greater than found in their marine relatives. This provokes an 
extended exposure time to different concentrations of pesticides, which depends on the 
exposure of females during gonad development and pesticide concentration in the 
ovaries. 

Because of the osmotic stress that freshwater environments present, freshwater decapods 
possess a thicker chorion for protecting embryos from external aggressions. This chorion 
also protects them from biocides, making eggs as resistant as adults in some freshwater 
prawns and crabs, leaving juveniles as the most vulnerable (Key et al., 2003; Li et al., 
2006). When the embryo is close to hatching, the chorion narrows to allow embryos to 
hatch, also allowing external agents to come into contact with embryos, making them 
more vulnerable to external agents, as observed in the prawn, Palaemonetes argentinus 
(Ituarte et al., 2005). 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 213 

Pesticides 



Effects /? <\ Effects 

Decrease in mobility, Less ability to feed 

flee capacity and ability l«i ability to courtine 

to find shelters or defense anc j mating 

thtmivtvM 



Man pr«dation Lwi eviierous famjlss 

Lass offspring 



\ / 

Decrease in Population 

Fig.6. Pesticide effects in crustaceans' behavior and its results in population. Crustacean 
image modified from Collins et al. 2004. 

The eggs of ovigerous females that live in contaminated areas may be resistant to pollutants, 
but toxicants may cause the death of juveniles after hatching, when they are not protected 
by a chorion. Moreover, moult events are a critical period for crustaceans, as their 
exoskeletons become softer and they are more vulnerable to external contaminants such as 
pesticides. In juveniles, the intermoult period is short, and the lethal effects of pesticides are 
increased during that critical period. 

4. Growth 

Growth is an interesting aspect in decapods in that it includes both internal and external 
factors. The intermoult period and increase in size are affected by different factors, such as 
diet (mainly protein, and lipid level variation), interspecific interactions (searching for 
agonistic behaviour and hierarchical conditions), temperature, biocides (or xenobiotic 
elements). Moreover, the growth in many species shows isometry and/ or allometry 
variations in the ontogeny, and thus growth pattern can be affected. The study methods are 
different according to a study's objectives. In some cases, the animals are evaluated in 
groups, e.g., with diets; in other cases, a study is conducted with isolated animals to observe 
the xenobiotic effects on growth through chronic assays. 

The capacity of an organism for survival, growth, and reproduction involves competition 
for energy resources at the individual level (Schmidt-Nielsen, 1997). Toxicant-induced shifts 
in energy allocations to these life-history activities will have important consequences on 
population. For example, higher respiration rates of estuarine crustaceans sublethally 
exposed to a variety of pesticides reduced juvenile growth by lowering growth efficiency 
rates, suggesting that increased metabolic demands lowered the amount of assimilated 
energy available for production of new tissue (McKenney & Hamaker, 1984; McKenney & 
Matthews, 1990). The assessment of changes in growth and energy stores of toxicant- 
sensitive life stages have a direct link to ecological consequences of environmental stress 
and can be useful as biomarkers to diagnose early damage in aquatic populations (Newman 
& Unger, 2003). 



214 Pesticides in the Modern World - Risks and Benefits 

Crustaceans do not grow continuously but by periodically shedding the hard exoskeleton in 
a process called moult or ecdysis. Moulting is a very important physiological process 
because it not only allows for growth and development of these animals, which possess a 
rigid, confining exoskeleton but is also tied to metamorphosis during the early stages of the 
life cycle and reproduction during the adult stage (Passano, 1960). The process of ecdysis of 
decapod crustaceans is an antagonistic interaction by ecdysone and the MIH (moult 
inhibiting hormone), which originates from the Y-organ and X-organ/ sinus gland (XO/SG) 
complex, respectively. The X-organ/ sinus gland complex is located within the eyestalks. A 
reduction in MIH in the haemolymph is believed to induce moulting and stimulate the Y- 
organs to synthesise and secrete ecdysone, which will be converted to the active moulting 
hormone 20-HE (20-Hydroxyecdysone). Moreover, a significantly lower level of 20-HE was 
recorded in the haemolymph during the interval of moulting (Chang, 1995). 
Limb regeneration is also an aspect of moulting. In this case, the regenerate first develops as 
a limb bud folded within a layer of cuticle and becomes free to unfold when the individual 
undergoes ecdysis as part of the moulting process (Fingerman et al., 1998). However, low 
levels of pollutants (such as chlorinated compounds) had an inhibitory effect on moulting 
and limb regeneration in some decapods (Fingerman ,1985). 

Growth rate is usually described in terms of independent moult periods. These consist of a 
description of the size increment for each individual moult (moult increment) and a 
description of the time increment between moults (intermoult period) (Hartnoll, 1982). In 
many decapod species, growth alterations by toxicant may be caused by variations in the 
moult increment, but principally by changes in the intermoult duration. A reduction in 
growth by the lengthening of the intermoult period was observed in juvenile prawns, 
Palaemonetes argentinus, during the first moult cycles exposed to cypermethrin (Collins & 
Cappello, 2006) and to chlorpyrifos and endosulfan insecticides (Montagna & Collins, 2007). 
In contrast, this same freshwater prawn showed a shortening in the intermoult period with 
a reduction in the moult increment at the highest concentration of glyphosate tested (0.070 
ml H) (Montagna & Collins, 2005). These changes may involve perturbations to the X-organ 
and the sinus gland, which affect the production and storage of the inhibitory moult 
hormone or, more integrally, the neurohormonal system located in the eyestalks. Snyder & 
Mulder (2001) reported a delay in the onset of moulting of larvae of the lobster, Homarus 
americanus, exposed to heptachlor. This delay was correlated with both reduced levels of 
circulating ecdysteroids and increases of some P450-dependent detoxifying enzymes. 
Although it is known that 20-hydroxyecdysone itself can induce the expression of these 
enzymes, it is quite possible that this induction can also be produced by some toxicants. 

5. Biocide effects on behaviour 

Among the movements made by an animal, there are vital movements, such as breathing 
and cardiac movements; locomotive movements for prey finding and predator escaping; 
and behavioural movements, such as courtship and copulation. Every movement, even the 
simplest, depends on the harmony of every single movement to complete a desired action, 
i.e., for swimming, a prawn needs each pleopod to move in the right direction at the right 
time and with the right intensity to accomplish the final desired movement. Movements are 
transmitted through the nervous system and the synaptic gap by neurotransmitters, such as 
acetylcholine, while they are inhibited by enzymes, such as acetylcholinesterase, which 
stops the nerve impulse. 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 215 

Some pesticides are acetylcholinesterase inhibitors in crustaceans and other animals 
(Saravana Bhavan & Geraldine, 2001; Braga da Fonseca et al., 2008). The inhibition of this 
enzyme enhances the contraction of skeletal muscles and impairs movement. When exposed 
to biocides that provoke an acetylcholinesterase inhibition, decapods are affected in their 
vital, locomotive and behavioural actions, with several different implications for the 
individual and the community. A prawn, crab or crayfish that is not able to swim or run 
correctly will be more susceptible to predation. Freshwater prawns and crabs exposed to 
acetylcholinesterase inhibitor biocides had a stimulus that improves appendage movements. 
Nevertheless, these movements are not synchronised, and the total movement efficiency is 
lower than that of normal locomotion. Prawn jumps are uncontrolled, and they keep 
jumping in the same place, without escaping from the area; crabs walking becomes frenetic, 
and they jump and walk, but move more slowly than normal. The increasing of impaired 
movements, which provokes a greater demand on muscle activity, more rapidly tires the 
affected animals. After the initial excitation, animals become quiet because of this tiredness, 
with slower movements and even immobility, making them more susceptible to predation 
(Williner & Collins, 2003; Collins et al, 2004; Collins & Cappello, 2006; Montagna & Collins, 
2008). In a natural environment, escape from natural predators will be more difficult if 
crustaceans are affected by this kind of biocide, enhancing predation and decreasing the 
population. 

Moreover, impaired movements not only affect locomotion as a way of escaping from the 
risk area but also affect the capacity of crustaceans to quickly locate refuges. Some 
freshwater crabs are pleustonic; they live between the roots of aquatic plants. As these roots 
act as filters for suspended organic matter and planktonic organisms, crabs go to the 
periphery for feeding. When detecting predators, they quickly migrate to the inside of the 
roots or stay still as a way of camouflage. Prawns and crabs also use rocks or burrows as 
refuges, either made by themselves or by other animals, and they swim or run to these 
refuges or bury themselves when they detect predators. Some crabs, especially the bigger 
species, use their chelipeds to attack their predators as a way of intimidating them and 
allowing themselves to flee (Collins et al. 2006, Collins et al., 2007). All these actions require 
a complex sequence of movements. If these decapods are affected by biocides, 
uncoordinated movements or tiredness will hinder their ability to find refuges, leading to 
increased predation and decreasing the population (Fig. 7). 

Coordination of movements is not only necessary for escaping predators but also for 
finding food resources. Freshwater crabs and prawns are omnivorous animals. Some 
groups are specialised to filter sand and clay, feeding on the microbiota inhabiting these 
sediments. Other groups eat algae, macrophytes and animals tissues. Animal food may 
come from carrion or from hunting live prey. Decapod prey includes insect larvae, 
cladocerans, copepods, benthic organisms such as annelids and molluscs, fishes, other 
crustaceans and even eggs, juveniles and adults of the same species (Collins et al., 2006, 
Collins et al., 2007). 

The hunting of mobile prey, such as fishes and crustaceans, and the manipulation of 
molluscs, which enclose themselves in their shells, requires both coordination in movements 
and strength. These actions become more difficult if decapods are subjected to 
acetylcholinesterase inhibitors or narcotic pesticides, decreasing the feeding capacity. 
Combining this decreased feed capacity with the increase in the energetic expenditure 
provoked by the impaired movements, biocide exposure eventually causes a depletion in 



216 Pesticides in the Modern World - Risks and Benefits 

energetic resources, with several detrimental results for survival, growth, gonad 
development and reproduction (Saravana Bhavan & Geraldine, 1997). 

The coordination of movements is also important in behaviours, such as territorial defence, 
courtship, mating and copulation. Decapod crustaceans, like many others animal species, 
have a courtship routine that is more or less complex, depending on the species. Mate 
selection is related to size and previous learning, and some crab species have a kind of 
"aggressive" courtship during which the male subjugates to the female (Fig. 7). 
Agonistic behaviour is common in decapods, especially in crabs, and it is characterised by a 
series of coordinated movements that lead disputes in which the animals involved are at 
risk of serious injuries, loss of pereiopods and/ or chelae or death of during combat. The 
more common resources involved in the disputes include shelters, mates and/or food. This 
behaviour may be affected by side effects of biocides; Williner & Collins (2003) and Collins 
& Cappello (2006), observed hyperactivity in freshwater crabs and prawns treated with 
cypermethrin. This hyperactivity capped oxygen consumption, resulting in an obligated 
hypoactivity during which there was a recovery state with reduced metabolism and lower 
oxygen consumption. This finding may show that decapods affected by biocides are 
acerbating the agonistic behaviour in the beginning, with a subsequent negative effect on 
recuperation. Reproductive behaviour may also be affected. Palaemonid males court 
females by swimming, chasing after them until they successfully place a piece of 
spermatophore on the female's abdomen, and cypermethrin produces erratic movements in 
Palaemonetes argentinus (Collins & Cappello, 2006). This could affect both courtship and 
reproduction itself, especially in regard to "freezing" of the spermatophore, as this requires 
coordination and precision. It is also possible that a female would find "defective" males 
under the influence of toxic and remove their sperm packages to obtain offspring with 
higher fitness or more viable eggs. It has been found that the effect of stress on egg masses 
affects the viability of these eggs (Siegel & Wenner, 1984). Stress may also disrupt or alter 
the chemical communication of these animals, as studies show that in many crustaceans, 
this type of communication occurs, permitting these animals to determine states of 
dominance. It is also known that during courtship, the chemical perception needed to 
recognise the state of female receptivity may also be disrupted by the action of biocides. In 
addition, in relation to energy, oxygen consumption increase as a result of biocide action 
causing hyperactivity, reduces the energy available for reproduction, either reducing the 
number of eggs or the effectiveness of the fertility of eggs (Siegel & Wenner, 1984), or in 
relation to the behaviour during parental care (Fig. 7). Moreover, shrimp in estuaries, such 
as penaeid shrimp, when exposed to biocides, exhibit decreases in the percentages of 
proteins as energy resources (Galindo Reyes et al., 1996). This alteration in energy storage 
could affect animals not only directly but the energy available for reproduction. Huang & 
Chen (2004) show that endocrine abnormalities were related to levels of testosterone and 
vitellogenin in Neocaridina denticulata treated with toxic. These abnormalities could affect the 
reproductive behaviour and gonadal development of these shrimp. It is known that female 
crabs, particularly freshwater crabs, incubate the eggs in their abdomens until hatching, and 
in some cases keep their offspring alive for some time after hatching, requiring sufficient 
energy to do so (Senkman, unpublished data). The effects of biocides may provoke the death 
of eggs and juveniles and the development of abnormal juvenile behaviour caused by stress. 
During embryonic development, many crustacean females move their eggs with opening 
and closing movements of the abdomen in rhythm, and their pleopods are used to remove 



Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 217 

bad eggs or foreign particles or microorganisms entering via the same motions for the 
abdomen. 

biocides 
oxygen consurnption^^^^^^^^^^ movements 



T 




decrease 

hipoactivity ^^^^" hiperactivity 

exacerbation ^^r 1 alteration retail Size 

agonistic behavior * reproductive behavior I affecting 



fitness decrease 



Fig. 7. Different effects that can occur when the animals are expose to biocide in relationship 
to oxygen consumption, activities, movements, and reproduction. All these affect the fitness 
of the species. 

Biocides are known to affect moulting events in some decapod crustaceans, affecting their 
growth and keeping many adult individuals in sizes below the average body size of 
conspecifics. It is estimated that the size of individuals is an important factor in mate choice, 
as there is a direct relationship in many crustaceans between adult size and the number of 
eggs a female is capable of carrying, so the effect of biocides may include the number of 
eggs, or indirectly the reduction of average adult size. 

6. Relationships between the external medium and an animal's body 

In assessing organisational levels, it is necessary to analyse those relationships beyond the 
physical dimension of the animal's body and that provoke the defined interactions. Among 
these relationships are the connections between the various components of a community, 
i.e., trophic webs. According to the environment, the trophic web may be more simple or 
complex, e.g., with more connections and interactions between components or with greater 
or lesser possibility of prey choice by top members. 

In these communities, whether they are subjected to fumigation or the biocides that enter 
the physical environment with runoff caused by rain, there will be species that are more 
sensitive than others, and these pollutants can make these species disappear or decrease 
their numbers extensively. This alteration will also be reflected in species that use this 
directly affected species as food, leading to increased competition among predators for 
fewer prey species. In this way, a decrease in diversity and a simplification of the system 
occurs. 

In addition, all community members are in contact with the biocide, which may accumulate 
in organisms. When predators eat contaminated prey the toxic conditions of the biocides 
from the lower elements of the food chain are transferred to the other trophic levels of the 
chain, magnifying their effects. 



21 8 Pesticides in the Modern World - Risks and Benefits 

Thus, the direction of flow of energy and matter through the food web can be affected, 
changing both direction and intensity, affecting the ability of each species and population to 
persist. 

The movement of populations occurs and can be induced by abiotic, biotic and human 
factors. In the last case, this movement can be induced after rainfall, when biocides 
accompany rainwater. The xenobiotics in sediment, suspended in colloids or dissolved in 
rainwater are trapped. These biocides can cause changes in abiotic conditions (pH, 
conductivity, nutrients) and water and sediment qualities. The different factors make it 
difficult to identify a cause and/ or cause-effect relationship, but these factors increase stress 
and impair various species' activities under various conditions. According to the timing of a 
rainfall event, the population may or may not be in its most vulnerable condition, based on 
its endogenous cycles (e.g., moult, reproduction). 

7. Conclusions 

In the modern world with the farmland activities, the aquatic communities are affected by 
different pesticides used in these agro-ecosystems. Herbicides, insecticides, fungicides are 
the most common elements used, and in some cases with several millions of liter what are 
used in the systems nearby to aquatic environments. This occurs due to the input of toxic 
compounds to water bodies by several ways, such as drift, and runoff provoking a risk to 
the fauna, and thus, creates the need of a constantly updates. The biological communities in 
rivers with floodplain and their tributaries are abundant, and with very high diversity. An 
interesting group is the decapods crustaceans by their abundance. These included prawns, 
crabs, pseudo-crabs and crayfish of South America. Populations of decapods could be 
reduced by lethal effects on the individual, and chronic alterations modify their fitness. The 
pesticide exposures cause damage in several tissues as hepatopancreas, gills, muscles and 
gonads, affecting the aquatic fauna. Among physiological functions, growth and 
reproduction could decrease by alterations in growth, gonad tissues, genetical material, and 
eggs development. Changes in metabolism, cell composition of the hepatopancreas, neuro- 
hormones have effect on behavior, growth rate, reproduction efficiency and survival due to 
the exposition of pesticide. Even more, these variations could affect the trophic web, and 
alter the transfers of material and energy into the aquatic systems. The proposed focus 
gives a snapshot from the macroscopic view of the ecosystem - community together to the 
molecular view. The different levels of organization with their temporal and spatial scale are 
necessary to achieve a better idea of the problem facing modern society with pesticides. 

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12 



Effects of Pesticides on Marine Bivalves: What 
Do We Know and What Do We Need to Know? 

T. Renault 

Ifremer/Laboratoire de Genetique et Pathologie, 

Ronce-les-Bains, 
France 

1. Introduction 

Estuaries are among the most productive environments in the world, by serving as feeding 
grounds, as nurseries for juvenile economically important fish and invertebrate larvae, and 
by providing shelter for many types of benthic organisms. However, they also rank among 
the most contaminated areas. 

Among pollutants, pesticides have become more common in estuarine areas. They are 
mainly introduced into rivers via run-off and then may enter marine areas, particularly 
estuarine and coastal zones. These pollutants may have major ecological consequences and 
could endanger organismal growth, reproduction or survival (Banerjee et al., 1996). 
Among important organisms inhabiting estuarine zones, bivalves are sessile and filter- 
feeder species, able to accumulate contaminants in their tissues. Moreover, bivalve farming 
is an ancestral activity all around the world. It has been expanded and intensified in the last 
century and represents a major economic activity in various countries. In the majority of 
cases, bivalve species are reared in estuaryine zones, continually impacted by pollutants 
including pesticides. Natural and man-made toxicants enter marine ecosystems by various 
routes, including direct discharge, land run-off, atmospheric deposition, in situ production, 
abiotic and biotic movements and food-chain transfer. 

Pollutant run-off into the ocean represents a potential threat to marine organisms, especially 
bivalves living in coastal environments. In this context, bivalve molluscs such as mussels 
and oysters have been postulated as ideal indicator organisms because of their wide 
geographical distribution, and sensitivity to environmental pollutants. They filter large 
volumes of seawater and may therefore accumulate and concentrate contaminants within 
their tissues (Ramu et al., 2007; Bernal-Hernandez et al., 2010). As an example, the level and 
extent of organic contaminants along the Korean coast has been estimated through a mussel 
watch program (Choi et al., 2010). Moreover, development of techniques allowing effect 
analysis of pollutant on bivalve biology may lead to the development of diagnosis tools 
adapted to analyze pollutant transfer towards estuarine areas. 

A pesticide is defined as a chemical substance used for killing pests, as insects, weeds or 
rodents. Pesticids are often classified by the type of organism: fungicides, herbicides, 
insecticides, nematocides and rodenticides. They are used especially in agriculture and 
around areas where humans live. Some are harmful to humans, either from direct contact or 



228 Pesticides in the Modern World - Risks and Benefits 

as residue on food, or are harmful to the environment because of their high toxicity, such as 
DDT (which is now banned in many countries). All pesticides act by interfering with the 
target species normal metabolism. Some inadvertently may affect other organisms in the 
environment, either directly by their toxic effects or via elimination of the target organism. 
By World War II, only about 30 pesticides existed. Dichloro-Diphenyl-Trichloro-ethane 
(DDT) was recognized as an insecticide until 1942. Other pesticides soon followed, such as 
chlordane and endrin. Poison gas research in Germany yielded the organophosphorus 
compounds, the best known of which is parathion. Further research yielded hundreds of 
organophosphorus compounds including malathion. The Environmental Protection Agency 
(EPA) estimates that the use of pesticides doubled between 1960 and 1980 with over 1.8 
billion kilograms a year used today worldwide. In most countries, pesticides must be 
approved for sale and use by a government agency. However pesticide regulations differ 
from country to country. To deal with inconsistencies in regulations among countries, an 
International Code of Conduct on the Distribution and Use of Pesticides has been adopted 
in 1985 under the umbrella of the United Nations Food and Agriculture Organization and 
then updated several times. 

Bivalves in culture may be affected by the presence of pesticides, potentially increasing their 
susceptibility to a wide range of infectious diseases. The effects of environmental 
contaminants may result from direct toxic actions on tissues or cells or from alterations of 
the homeostatic mechanisms including the immune system (Coles and Pipe, 1994; 
Carajaville et al., 1996). It has been suggested that bivalves may be weakened in relation to 
the presence of these pollutants. It has been shown in several vertebrates and invertebrates 
that pesticides are capable of diminishing immune defenses and/ or of modifying genomes. 
They may render animals more vulnerable to infectious diseases (Ross et al., 1996; Gagnaire 
et al., 2007). 

Although pesticide effects on marine bivalves have been already studied in bivalves, a few 
of reviews summarizing their different effects are available. In this context, one of the major 
objectives of this chapter relies on summarizing existing body of data on pesticide detection 
in marine environments and their effects on bivalve physiology including genotoxicity and 
immunotoxicity. Moreover, another aim of the present chapter is to identify the topics on 
which scientific data are needed in order to better understand the complex interactions 
between pesticides, environment, marine bivalves and their infectious agents. 

2. Pesticides in the marine environment 

Aquatic habitats are particularly subjected to contamination by pesticides, via run-off, 

leaching, spray drift or accidental spills. Pestices contamination of the marine environment 

has been and is monitored worldwide through analysis of water, sediment and marine 

species samples in order to elucidate the contamination status, distribution and possible 

pollution sources and to assess the risks on aquatic organisms and human. 

Levels of pesticides measured in superficial waters generally range below lethal 

concentrations for aquatic species. However, sublethal adverse effects may result from 

exposure to these products at environmentally relevant concentrations. 

Buisson et al. (2008) reported recently results about the monitoring of contamination levels in 

the Pacific cupped oyster, Crassostrea gigas, reared in Normandy (France). Six herbicides were 

detected in seawater for a total of 15 herbicides. Although the most estuarine sites showed 



Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know? 229 

relatively high values in sea water samples, no pesticides were detected in the flesh of 
collected oysters (Buisson et al. 2008). At the contrary, Monirith et al. (2003) reported that all 
samples collected from all the sampling sites demonstrated the detection of organochlorines 
(OCs) with considerable residue levels of p,p(')-DDT and alpha-hexachlorocyclohexane (HCH) 
in mussels collected from coastal waters in the Asia-Pacific region. 

Pandit et al. (2006) conducted a multi-compartment monitoring (sediment, water and 
marine species) of residue levels of pesticides in coastal marine environment of Mumbai in 
India. The total HCH concentration in sediment samples varied from 3.8 to 16.2 ng g- 1 
lindane (gamma-HCH) contributing almost 55% to the total HCH. The concentration of total 
HCHs in seawater ranged from 0.16 to 15.92 ng L- 1 and concentrations of total DDT varied 
from 3.01 to 33.21 ng IA 

The presence of herbicides, such as diuron, has been also detected in many aquatic 
ecosystems worldwide. For instance, in France, diuron has been detected in surface waters 
with concentrations ranging from 0.05 ng L- 1 to 20.3 jig L- 1 (Leonard, 2002). In Atlantic bays 
and estuaries, concentrations up to 0.7 and 1 ng L- 1 have been reported (Munaron et al., 
2006). 

Due to its toxicity, the use of diuron has been forbidden by French policies since 2008. 
Diuron and isoproturon are also included in the list of priority to contaminants of the EU 
Water Framework Directive (European Co mission, 2000). However, it is well-known that 
some herbicides may persist in the environment even if their use has been banned, e.g. 
atrazine (EEA, 2000). A recent study reported the presence of diuron on French aquatic 
environments, confirming its persistence despite restriction policies (Pesce et al., 2010). 
Diuron metabolites such as DCPU (N-(3,4 dichlorophenyl)-urea), DCPMU (N-(3,4 
dichlorophenyl)-N-(methyl)-urea) and DCA (3,4-dichloroaniline) have also been detected in 
aquatic environments (Munaron et al., 2006). Studies on biofilms have reported DCPMU to 
be more toxic than DCA (Pesce et al., 2010). However, the principal product of degradation 
of diuron reported in the literature is DCA, which has shown to be more toxic for various 
organisms of higher trophic levels, such as crustacean, insects and fish (Giacomazzi & 
Cochet, 2004). This product exhibits higher toxic effects than the parent diuron, and can 
affect organisms, such as crustacean with low concentrations (1 ng. -1 , Giacomazzi and 
Cochet, 2004). 

Different compounds including herbicides and their metabolites (Lanyi & Dinya, 2003; 
Sorensen et al., 2003; Vargha et al., 2005) are detected simultaneously in aquatic 
environments, suggesting that experimental approaches with toxicant mixtures are needed. 
Studies with diuron and its metabolites have shown additive, enhanced, antagonistic or 
independent effects (Knauert et al., 2008; Pesce et al., 2010; Neuwoehner et al, 2010). Thus, 
there is still a lack of data concerning the toxicity and effects of pesticide metabolites on 
bivalves, whether individually or in mixture with their parent compounds. 

3. Lethal effect of pesticides on marine bivalves 

Several studies have been conducted in various marine bivalve species in order to define 
LCsofor different pesticides including DDT, diuron, atrazine or lindane. 
Chung et al. (2007) evaluated the sensitivity of the juvenile hard clam, Mercenaria mercenaria, 
to DDT (organochlorine pesticide) by exposure to contaminated sediments (10 day) and 
seawater (24-h). The aqueous LC50 (24h) value was defined at 0.61 mg L- 1 DDT. and the LC50 
(10 day) value for sediment toxicity tests was 5.8 mg kg- 1 DDT. The authors concluded that 



230 Pesticides in the Modern World - Risks and Benefits 

based on comparisons to toxicity data for other marine species, the hard clam, Mercenaria 

mercenaria, is one of the more sensitive species to contaminants. 

No significant mortalities were reported after two months of exposition to 100 mg l/ 1 of 

diuron while with 100 jig L^oi isoproturon, 60% of mortalitites were observed (Moraga & 

Tanguy, 2000). Isoproturon has shown to be present at lower concentrations than diuron on 

aquatic environments (Munaron, 2006). 

Lawton et al. (2010) studied the effects of atrazine on the hard clam, Mercenaria mercenaria, in 

aqueous and sediment laboratory assays. Through an acute aqueous bio-assay, these 

authors determined a 96h LC50 for the juvenile clams at 5608 jig L 1 . They conducted also a 

chronic aqueous bio-assay at low atrazine concentrations and a chronic sediment bioassay 

over a 10 day exposure period to examine both lethal and sublethal (dry mass, shell size, 

and condition index) endpoints (Lawton et al., 2010). Qn the basis of their results, the 

authors suggested that atrazine is not directly toxic to M. mercenaria at environmentally 

relevant concentrations. 

Bouilly et al. (2003) reported similar results for the Pacific cupped oyster, Crassostrea gigas:, 

in adult and juvenile animals subjected to 2 different concentrations of atrazine (46.5 nM 

and 465 nM). These authors did not observed any effect on mortality. 

In vivo in laboratory assays testing 10 different concentrations (0 to 10 mg L _1 ) of lindane 

(gamma-hexachlorocyclohexane [gamma-HCH]) allowed to define the median lethal 

concentration (LC50) after a 12 day period as 2.22 mg l/ 1 in the Pacific cupped oyster, 

Crassostrea gigas (Anguiano et al., 2006). Lindane and isoproturon tested at concentrations of 

up to 10 mg L" 1 for a 9 day esposure period showed negative effects on survival and growth 

of Pacific cupped oyster, Crassostrea gigas, larvae (Hiss & Seaman, 1993). 

Domart-Coulon et al. (2002) assessed the acute cytotoxicity of an organic molluscicide, 

Mexel-432, used in antibiofouling treatments in industrial cooling water systems on primary 

cell cultures derived from 2 marine bivalve species, the Pacific cupped oyster, Crassostrea 

gigas, and the carpet clam, Ruditapes decussatus. 

4. Genotoxicity in marine bivalves 

Results reported by Jha et al. (2002) suggested that tributyltin oxide is both cytotoxic 
(proliferation rate index) and genotoxic (sister chromatid exchanges and chromosomal 
aberrations) to embryo-larval stages in the blue mussel, Mytilus edulis. 

Bouilly et al. (2003) researched potential genotoxic effects of atrazine in the Pacific cupped 
oyster, Crassostrea gigas. Adult and juvenile oysters were subjected to 2 concentrations of 
atrazine: 46.5 nM, representing a realistic potential exposure (peak value found in polluted 
environment) and 465 nM. These authors reported significant differences in aneuploidy 
after atrazine treatments in comparion to control: 9% in control oysters, 16% at 46.5 nM and 
20% at 465 nM atrazine. Similar aneuploidy levels were observed in adults and juveniles. 
Bouilly et al. (2007) showed that the herbicide diuron induced also aneuploidy in adult 
Pacific cupped oysters after a 11 week exposure period at 300 ng L- 1 and 3 jig L- 1 . The 
induced aneuploidy observed appeared to be transmitted to the next generation as offspring 
exhibited significantly higher aneuploidy levels when their parents had been exposed to 
diuron (Bouilly et al., 2007). 

Genotoxicity induced by lindane at 0.7 mg L- 1 was also demonstrated in Pacific oyster, 
Crassostrea gigas, hemocytes after a 12 day contamination period (Anguiano et al., 2006). 



Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know? 231 

Wessel et al. (2007) investigatied embryotoxic and genotoxic effects of the organochlorine 

pesticide, endosulfan, on Crassostrea gigas embryos. Embryotoxicity and genotoxicity in 

terms of DNA strand breaks were observed for 300 nM and 150 nM. 

Siu et al. (2008) used green-lipped mussels (Perna viridis) in order to study the 

bioaccumulation of organic pollutants, including organochlorine pesticides. Micronuclei and 

DNA strand breaks were observed in mussels transplanted in different sites and collected 

after 4, 8, 12, 16 and 30 days. 

Revankar and Shyama (2009) explored genotoxic effects of monocrotophos, an 

organophosphorous pesticide, at different time periods, 2, 3, 7 and 14 days. A significant 

increase of micronuclei in a dose dependant manner was observed indicaring possible 

chromosomal damages induced by monocrotophos. 

5. Immunotoxicity in marine bivalves and susceptibilty to infectious diseases 

The impact of contaminants and other environmental factors on the immune system of 
bivalves is an issue of ecological and economical concern, because it may result in clinical 
pathology and disease, by increasing the susceptibility of affected organisms to pathogens. 
Contaminants known to induce alterations of immune functions including pesticides (Vial et 
al., 1996; Banerjee et al., 1996; Banerjee et al., 2001) are present in almost all coastal areas. 
Among physiological processes possibly disturbed by pollutants, the immune system is 
likely to be one of the more sensitive (Baier-Anderson & Anderson, 2000; Fournier et al., 
2000). 

In contrast to the vertebrate immune system which consists of innate and acquired 
mechanisms, invertebrate immunity relies only on innate defence mechanisms. The fact that 
invertebrates represent more than 90% of the total number of species living on earth 
demonstrates the efficiency of their «primitive» host defence systems. It becomes more and 
more obvious that some of these innate mechanisms are conserved in invertebrates and 
vertebrates (Medzhitov et al., 1997; Means et al., 2000). Thus, the fundamental importance of 
the toxically-induced modulation of non-specific immune functions has increasingly been 
perceived. 

Bivalve immunity is mainly supported by hemocytes and participate directly in eliminating 
pathogens by phagocytosis (Cheng, 1981; Feng, 1988). In addition, hemocytes produce 
compounds including lysosomal enzymes and antimicrobial molecules which contribute to 
the destruction of pathogens (Coles & Pipe, 1994). 

Investigating the effects of pesticides on hemocyte functions and immunity in bivalves has 
been based on the monitoring of several biomarkers (Pipe & Coles, 1995). As an example, 
Gagnaire et al. (2006) tested the effect of 23 pollutants on Pacific cupped oyster haemocytes 
by flow cytometry monitoring different cell parameters and demonstrated that 3 pesticides 
(2,4D, paraoxon, and chlorothalonil) induced a modulation of hemocyte activities. However, 
biomarkers used differ very often between published studies. 

Triforine, a fungicide, induced decreased hemocyte viability in the eastern oyster, 
Crassostrea virginica (Alvarez & Friedl, 1992). Cytotoxic effects were also observed in adult 
Pacific cupped oyster, C. gigas, hemocytes: the mean cell viability was significantly 
decreased at 1.0 mg L- 1 of lindane (gamma-hexachlorocyclohexane) after 12 day exposure 
period (Anguiano et al., 2006). Alteration in cell viability was also reported in the blue 
mussel, Mytilus edulis, exposed to 0.1 mg L- 1 azamethiphos, an organophosphate pesticide 



232 Pesticides in the Modern World - Risks and Benefits 

(Cantry et al., 2007). Moreover, a mix of herbicides containing atrazine, diuron and 
isoproturon showed an effect on C. gigas hemocyte aggregation (Auffret et Oubella., 1997). 
Chlordan, an insecticide, demonstrated effects on C. virginica hemocyte phagocytosis at 250 
(iM in vitro (Larson et al., 1989). A decreased phagocytosis activity was observed after a 
triforine exposure in the eastern oyster, C. virginica (Alvarez and Friedl, 1992). A pesticide 
mixture (alachor, metolachlor, terbutylazine, glyphosate, diuron, atrazine, carbaryl and 
fosteyl aluminium) representative for surface waters of the Marennes-Oleron Basin 
(Charente Maritime, France, 0.25 nM to 4 nM) induced a decrease of phagocytic activity 
(Gagnaire et al., 2007). Moreover, Cantry et al. (2007) reported a decrease in phagocytic 
index in the blue mussel, Mytilus edulis, after a short exposure to 0.1 mg I/ 1 azamethiphos. 
This result suggests that azamethiphos can modulate haemocyte function in mussels at 
environmentally relevant concentrations. 

At the contrary, Gagnaire et al. (2003) reported no effect on cell viability, cell cycle and 
cellular activities except for peroxidase activity for Pacific cupped oyster haemocytes 
exposed to atrazine in in vitro and in vivo assays. 

Pentachlorophenol decreased the production of ROS by the inhibition of NADPH 
production in the eastern oyster, Crassostrea virginica (Baier-Anderson & Anderson, 1996). 
Dieldrin, tested in vitro on C. virginica hemocytes induced a decrease of chemiluminescence 
at concentrations ranging from 3 to 300 jiM (Larson et al., 1989). Hemocytes of C. virginica 
exposed to chlorothalonil (fungicide) for 20 h at concentrations between 4 nM and 2 jiM 
showed no modification of cell mortality and phagocytosis, but a decrease of ROS 
production (Baier-Anderson & Anderson, 2000). 

In the past decades, the emergence of infectious diseases has been reported in marine 
species and disease outbreaks have also increased (Harvell et al., 1999). According to 
Snieszko (Snieszko, 1974), the development of an infectious disease results from an 
unbalance between the host and the pathogen due to external factors (including pollutants) 
and/or internal factors of both protagonists (virulence of the pathogen, susceptibility of the 
host). Animals presenting impaired defence mechanisms may be more susceptible to 
infectious diseases. 

Demonstration of the relationship between pollution and increase of susceptibility to 
infectious diseases exist in vertebrates (Fournier et al., 1988; Van Levoren et al., 2000; Jepson 
et al., 2005), a few of studies was carried out in invertebrates (Galloway & Depledge, 2001). 
Rare studies have attempted to link contaminant presence and susceptibility to infectious 
diseases in marine molluscs and demonstrated harmful effects of pollutants in bivalves. 
Contamination of the eastern oyster, Crassostrea virginica, by polluted sediment and 
tributyltin increased the intensity of Perkinsus marinus infection, but no cellular or humoral 
parameters were modulated (Anderson et al., 1996; Chu et al., 2002). Anderson et al. (1981) 
demonstrated previously that the hard clam, Mercenaria mercenaria, exposed to PCP were 
unable to kill injected bacteria. Kim et al. (2008) studied the relationship of parasite detection 
to contaminant body burden in sentinel bivalves through a 'Mussel Watch 1 Program. These 
authors showed that correlations between parasites/ pathologies and pesticides were 
frequent in mussels and oysters (Kim et al., 2008). 

The Pacific cupped oyster, Crassostrea gigas, has been also used to evaluate the impact of a 
pesticide mixture (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, 
terbuthylazine, diuron and carbaryl) on some immune-related parameters and to 
demonstrate a relationship between infectious diseases, defence capacities and pollutants. 



Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know? 233 

Indeed, a mixture of 8 pesticides reduced phagocytosis on hemocytes and enhanced 
susceptibility to Vibrio splendidus (Gagnaire et al., 2007). Pacific cupped ysters were exposed 
over a 7 day period to the mixture of pesticides. The pesticides were selected on the basis of 
spread amounts in the Marennes-Oleron Basin (Charente-Maritime, France), one of the most 
important oyster producing areas in France (Leonard, 2002; Munaron et al., 2006). Moreover, 
a down-regulation of the LBPB/BPI, TIMP and lysozyme genes were reported in Pacific 
oysters exposed to the mixture of 8 pesticides (Gagnaire et al., 2007). 

6. Other effects of pesticides on marine bivalves 

The evaluation of acetylcholinesterase activity in marine organisms has been and is at 
present time extensively used as a biomarker of exposure to neurotoxic agents such as 
organophosphorus and carbamate pesticides. Indeed, organophosphorous compounds and 
carbamates including paraoxon and carbaryl are known to inhibit acetylcholinesterase 
(AChE) and carboxylesterase (CE) (Cooreman et al., 1993). 

Paraoxon inhibited the activity of AChE in the hepatopancreas of the blue mussel, Mytilus 
edulis, in vitro at concentrations ranging from 1 jiM to 1 mM (Ozretic and Krajnovic-Ozretic, 
1992). Inhibition by carbaryl was less distinct. AChE fromM. edulis hemocytes was inhibited 
in vitro by 0.1-3 mM paraoxon, eserine and DFP (Galloway et al, 2002). Cantry et al. (2007) 
showed that exposure of the blue mussel, M. edulis, to 0.1 mg L- 1 azamethiphos, an 
organophosphate pesticide used to combat sea lice infestations in farmed salmonids, for 
periods of up to 24h caused a significant reduction in acetylcholinesterase activity in both 
the haemolymph and the gill. However, cholinesterases found in the Pacific cupped oyster, 
Crassostrea gigas, appeared to be insensitive to organophosphorous insecticides (Bocquene et 
al.,1997). 

Anguiano et al. (2006) showed that after a 4 h exposure to lindane (gamma- 
hexachlorocyclohexane), filtration rates of adult Pacific cupped oysters, Crassostrea gigas, 
were significantly reduced compared with controls at concentrations of 0.3 and 0.7 mg IA 
However, a short term exposure of the blue mussel, Mytilus edulis, to azamethiphos did not 
change the feeding rate (Chantry et al., 2007). Studies carried out in adult Pacific cupped 
oysters revealed that diuron induces partial spawning and atrophy of the digestive 
epithelium after 1 week of exposure at 1 \ig L" 1 (Buisson et al., 2008). 

Greco et al. (2010) investigated effects of a mixture of herbicides on the physiological status 
of the soft clam, Mya arenaria. Clams were exposed for 28 days to 0.01 mg L- 1 of a pesticide 
mixture: dichlorophenoxyacetic acid (2,4-D), 2-(2-methyl-4-chlorophenoxy) propionic acid 
(mecoprop), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Although a gradual sexual 
maturation was reported in both sexes during the course of the experiment, females 
demonstrated a higher sensitivity to pesticides compared to males. 

Favret and Lynn (2010) during the course of a study monitoring sperm viability by flow 
cytometry in the eastern oyster, Crassostrea virginica, after exposure to a pesticide 
(Bayluscide) reported effects on mitochondrial membrane potential and plasma membrane 
in the sperm. Buisson et al. (2008) studied impact of pesticides in the cupped Pacific oyster, 
C. gigas, and reported partial spawning and atrophy of the digestive tubule epithelium in 
relation to pesticides. 

A study with a mix of herbicides containing atrazine, diuron and isoproturon revealed 
effects on gene expression in the Pacific cupped oyster, Crassostrea gigas (Tanguy et al., 
2005). Gagnaire et al. (2007) studied also the impact of pesticides on C. gigas, monitoring 



234 Pesticides in the Modern World - Risks and Benefits 

gene expression in hemocytes by real-time PCR. The expression of genes involved in C. gigas 
hemocyte functions was up-regulated in pesticide-treated oysters compared to untreated 
oysters after a bacterial challenge. The authors hypothesized that gene over-expression 
could lead to an injury of host tissues, resulting in higher mortality rates. 
Collin et al. (2011) explored under experimental conditions the effects of a cocktail of three 
pesticides (lindane, metolachlor and carbofuran) on physiological functions of the Pacific 
cupped oyster, C. gigas, using the suppression subtractive hybridisation technique. The 
authors reported a site and organ-specific response to the pesticides. Effects of imidacloprid 
and thiacloprid, 2 neonicotinoid insecticides, at transcriptomic and proteomic levels in the 
marine mussel, Mytilus galloprovincialis, were also reported by Dondero et al. (2010). 
Tlili et al. (2010) compared the size-distribution of the intra-sedimentary bivalve Donax 
trunculus collected in two sites in Tunisia, a polluted site and a comparatively reference site 
The auhors showed that the size-distribution from the polluted site consisted of 4 
cohorts.whereras 5 cohorts were observed in the comparatively reference site. Moreover, the 
mean total length size and the growth rate of cohorts were significantly reduced in the 
impacted site compared to the reference site. These results suggest effects of pollutants on 
marine bivalves at a population level with an ecological relevance. 

Pariseau et al. (2010) studied haemic neoplasia in the soft-shell clam Mya arenaria, in relation 
to exposure to fungicides, chlorothalonil and mancozeb, without demonstrating a link. 

7. Conclusions and perspectives 

The results obtained through the cited studies alerts to the negative effects of pesticides in 
bivalves and may be important to initiate and implement programs to protect the bivalve 
estuaries. These studies bring scientific evidence regarding the biological effects of 
pesticides on the animals inhabiting contaminated estuaries and the potential effect of the 
contaminates on shellfish and on human health if the seafood is consumed. 
The great variability of response (depending on duration of exposure, toxicant 
concentration, test species or experimental conditions) is a reminder that the effects of 
pollutants on the marine environment cannot be assessed by simple methods (e.g. short- 
term bioassays with one or two test species). As an example, Greco et al. (2010) investigating 
effects of a mixture of herbicides on the physiological status of the soft clam, Mya arenaria, 
showed that in clams kept at 18 C C, pesticides appeared to induce minor effects compared 
with animals kept at 7°C. They concluded that increased temperature may modify the 
response of Mya arenaria to pesticides. 

It is recognized that bivalve habitats may differ in environmental parameters. Thus, animals 
may be exposed to numerous variables that include other pollutants, different temperatures, 
salinities, amounts of dissolved oxygen, and changes in pH. In this context, a better 
understanding of the possible interactions between pesticides and other abiotic 
environmental factors (temperature, salinity,) and biotic factors associated with the 
physiological status of bivalves is necessary. 

As it is possible to evaluate only a limited number of environmental factors in laboratory 
assays, it appears difficult to investigate all of the potential environmental factors that also 
may affect bivalve physiology. 

A lot of studies concerning effects of pesticides in bivalves have been carried out using high 
pollutant concentrations. However, levels of pesticides measured in superficial waters 
generally range below lethal concentrations for aquatic species. In this context, sub-lethal 



Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know? 235 

adverse effects need to be more documented through experiments carried out using 
pesticides at environmentally relevant concentrations. 

Among studies focused on pesticides, most of them have been carried out by exposing 
animals to relatively long periods of time (Auffret et Oubella., 1997; Tanguy et al., 2005; 
Bouilly et al., 2007, Buisson et al., 2008) giving an insight on the effects of chronic exposures 
on physiological functions of the organism. 

Nevertheless, it is well known that in natural waters, uneven concentrations of pesticides 
are found in the water mass because of different factors such as seasonal agricultural 
practices, weathering processes and peak concentrations are often found in the aquatic 
environment for short periods of time (Munaron, 2006; Hyne et al., 2008). Thus, long-term 
studies may be not so predictive of what could happen on a natural environment. Short- 
term exposures of herbicides under laboratory controlled conditions have shown to exert an 
effect on aquatic organisms (Bretaud et al., 2000; Saglio et al., 2002). They can give an insight 
of the potential effect of contaminants in organisms in the natural environment. 
In order to assess the impact of persistent pollutants on the marine ecosystem a suite of 
biomarkers are being extensively used worldwide (Ozretic & Krajnovic-Ozretic, 1992; Lowe 
& Fossato, 2000). These biomarkers are being used to evaluate exposure of various species of 
sentinel marine organisms (e.g. mussels, clams, oysters.) to and the effect of various 
pesticides using different molecular approaches (Wong et al., 1992; Cajaraville et al., 1996; 
Galloway et al, 2002). 

As an example, Matozzo et al. (2010) developed a multibiomarker approach in order to 
assess effects of environmental contaminants in the Manila clam, Ruditapes philippinarum, 
collected in 8 sites of the Lagoon of Venice (Italy). The authors used several biomarkers 
includig total haemocyte count and lysozyme activity, acetylcholinesterase activity in gills, 
vitellogenin-like protein levels in both digestive gland and cell-free haemolymph, and 
survival-in-air widely used to evaluate general stress conditions. In addition, different 
pollutants were also measured in collected animals. Results showed that the selected 
integrated approach between biomarkers and chemical analyses is a useful tool in 
biomonitoring ( Matozzo et al., 2010). 

Different compounds including different pesticides have been found simultaneously in 
aquatic environments, underlining that experimental approaches with toxicant mixtures are 
needed. Most of the studies evoked before, have been carried out in adults, but juvenile 
organisms are known to be generally more sensitive to environmental stress than adults 
(Perdue et al., 1981). Additional residue-effects data on sublethal endpoints, early life 
stages, and a wider range of legacy and emergent contaminants will be needed. 
Finally, research in ecotoxicology needs also to fill the gap existing between sub-organismal 
responses to toxicants and effects occurring at higher levels of biological organisation (e.g. 
population) (Tlili et al., 2010). 

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13 



Immunotoxicological Effects of Environmental 
Contaminants in Teleost Fish Reared 

for Aquaculture 

Alberto Cuesta, Jose Meseguer and M. Angeles Esteban 
Fish Innate Immune System Group, Department of Cell Biology and Histology, 

Faculty of Biology, University ofMurcia, 

Spain 

1. Introduction 

Contamination is one of the major problems associated with the environmental sciences. 
Many of the environmental pollutants affect to the different aquatic animals to certain 
degree depending on the toxic substance, concentration, self-life and animal behaviour and 
biology. Direct ingestion of environmental contaminants and bioaccumulation of toxic 
substances in bivalves, crustaceans, molluscs or fish for human supply is a serious task to 
consider in human nutrition. Furthermore, it is known that to provide the necessary 
proteins that need and will need the world's population must intensify efforts in production 
of both proteins of plant origin and animal origin. Among the latter is predicted that 
aquaculture will be one of the fields over the coming years will increase. In this regard, 
aquaculture is trying for some decades to compensate this negative balance for human 
consumption. Among the important issues to consider in the aquaculture business the 
impact of the environmental contaminants in the species produced for humans need to be 
controlled by the farmer. In this specific field, most of studies have evaluated the toxic 
effects in terms of fish viability or induction of tumors using different fish models. However, 
relevant fish species for aquaculture are less used in these experiments. Moreover, the 
impact of the environmental contaminants in the immune response of these fish, and 
consequently in the disease resistance, have received much less attention. 

2. Overview of the teleost fish immune response 

Fish are the first group of vertebrate animals with both innate and adaptive immune 
responses and are essential for proper understanding of the immune system and its 
evolution. The fish adaptive immune responses are less effective than in mammals because 
they are poikilotherms and completely dependent on the environmental temperature. 
Therefore, the importance of the innate immune response is more relevant, but not 
exclusive, in the fish disease resistance to pathogens. Overall, the mechanisms and 
molecules involved in the immune response are quite well conserved during the immune 
system evolution. However, there are major differences in terms of haematopoietic organs 
structure and function as well as in leucocyte distribution and function (Figure 1). 



242 



Pesticides in the Modern World - Risks and Benefits 



Th, thymus 
Pr, pronephros 




Kid, mesonephros 
Spl, spleen 



B, lymphocyte B 

Tc, cytotoxic T lymphocyte 

Th, helper T lymphocyte 

MM, monocyte-macrophage 

G, granulocytes 

Ig, immunoglobulin 

TCR, T-cell receptor 

NCC, nonspecific cytotoxic cells 

NK, natural killer cells 



Humoral immunity 

(serum, mucus) 



Cellular immunity 

(kidney, spleen, thymus, blood, peritoneum) 



Antibodies 

Complement 

Lysozyme 

Lectins 

C-reactive protein 

Interferons 

Transferrin 

Anti-proteases 

Proteases 

Eicosanoids 

Cytokines 




CD8 




Circulating antibodies 

Antibody-forming cells 

Proliferation 



Specific cytotoxicity 
Proliferation 



Phagocytosis 

Respiratory burst 

Antigen presentation 



Cytotoxic activity (NCCs, NK, Tc), circulating leucocytes, infiltration, distribution, 
gene expression, cytokines 



Fig. 1. Fish immune system organization (from Manning, 1998) and representative humoral 
and cellular immune responses used in immunotoxicological studies. 

Firstly, the immune tissues are quite different since fish lack the bone marrow and 
lymphatic nodules (Manning, 1998). Thus, pronephros (anterior/ head-kidney) is the main 
lympho-haematopoietic tissue in fish, whilst the posterior part or mesonephros is mainly 
excretory and the first site for development and B cells production. Thymus is the main 
tissue for T cells development and maturation whilst spleen is the main secondary 
lymphoid tissue in fish. Other important site for the immune response is the mucosal 
associated-lymphoid tissue (MALT), disperse in the skin, gill and gut. The leucocyte-types 
present in fish are quite similar between vertebrates but with some specific differences 
(Meseguer et al., 1994; Secombes et al., 2005; Miller et al., 1998; Rombout et al., 2005). Thus, 
fish lymphocytes are responsible for the production of antibodies (B cells) and the specific 
cellular immune response (T cells). B lymphocytes express and secrete immunoglobulin M 
(IgM), respond to the mitogen lipopolysaccharide (LPS) and constitute about 30% of the 
circulating lymphocytes. T lymphocytes are mainly detected in the thymus, express the T- 
cell receptor (TCR) and proliferate with the mitogens concanavalin A and 
phytohemagglutinin (PHA). They are responsible for the humoral and cellular immune 
response against T-dependent antigens by the different populations of CD4+ (Th or helper) 
and CD8+ (Tc or cytotoxic). Moreover, there are also subpopulations of fish lymphocytes 
lacking proper cell markers, Ig or TCR, and constitute the natural killer (NK) cells (Shen et 
al., 2002). By other side, monocyte-macrophages are the leucocytes displaying similar 
characteristics to both mammalian circulating monocytes and tissular macrophages. 



Immunotoxicological Effects of Environmental 

Contaminants in Teleost Fish Reared for Aquaculture 243 

Moreover, they are mainly localized in kidney and spleen where they concentrate the 
ingested particles and aggregate in melano-macrophage (MM) centres. Granulocytes can be 
divided in neutrophils, eosinophils and basophils according to their staining properties but 
in the case of fish the distribution and functions do not fit well with their mammalian 
counterparts. Monocyte-macrophages and some granulocytes form the phagocytic cells 
involved in phagocytosis of particulated antigens and in production of a machinery of lytic 
enzymes and the respiratory burst reaction, in which very toxic reactive oxygen species 
(ROS) and nitrogen intermediates (RNI) are produced. Finally, nonspecific cytotoxic cells 
(NCCs) are involved in the lysis of tumor cells, virus-infected cells and parasites in a similar 
fashion than the mammalian NK cells (Evans et al., 1984). However, they are a 
heterogeneous population (lymphocytes, granulocytes and/ or monocyte-macrophages) and 
therefore some authors talk of nonspecific cytotoxic activity more than a cellular type or 
population (Cuesta et al., 1999). 

The humoral immune response is a compilation of proteins and glycoproteins with defense 
functions found in the fish plasma and other body fluids such as mucus or sexual products 
(Kaattari & Piganelli, 1997). The complement system, in plasma and mucus, shows classical, 
alternative and lectin activation pathways with levels 5-10 times higher than in mammalian 
species with most of its components detected and characterized (Holland & Lambris, 2002). 
Direct lytic activity against bacteria, virus and parasites is the most relevant and studied 
function but it also acts as opsonin, chemotactic and neutralize endotoxins (Boshra & 
Sunyer, 2006). An important bacteriolytic enzyme is the lysozyme, mainly found in eggs, 
mucus, plasma and leucocytes (Magnadottir, 2006). There are also other innate immune 
factors such as acute phase proteins (C-reactive protein CRP), antimicrobial peptides, 
interferon (IFN), lectins, proteases, protease inhibitors or eicosanoids (Secombes, 1996; 
Aranishi, 1999; Bayne & Gerwick, 2001; Robertsen, 2006; Cammarata et al., 2007; Cuesta et 
al., 2008a). Finally, and the most interesting in fish, Ig are the major component of the 
adaptive humoral immune response. Fish were thought to have only one immunoglobulin 
isoform, the IgM. The fish IgM is tetrameric instead of pentameric as it occurs in mammals. 
Both membrane and soluble forms are observed by alternative processing of the mRNA 
(Wilson et al., 1990). Igs are found in the membrane of the B lymphocytes and this can be 
used to separate Ig+ and Ig- cells. The Ig functions are antigen neutralization, precipitation, 
opsonization and activation of the classical pathway of the complement system. In the last 
years, the presence of other Ig isoforms (IgD, IgZ or IgT) is throwing some light into the 
repertoire of fish immunoglobulins and their evolution in vertebrates (Hsu et al., 2006; 
Hikima et al., 2011). 

3. Immunotoxicological effects of environmental contaminants 

Environmental contaminants are widely distributed in aquatic environments. Although 
many of them are prohibited or restricted most of them are very persistent in the nature. 
Field and semi-field experiments are good to have suspicions about the contaminant 
presence but the setup of laboratory experiments with controlled parameters and precise 
and pure compounds are strictly necessary to understand the impact on fish immune 
response and their potential mechanisms. In line with the immunotoxicological studies in 
mammals, most of fish studies have evaluated the immune response (Figure 1) by 
measuring the macrophage functions (i.e. phagocytosis and ROS production), 
lymphoproliferative responses, host disease resistance, antibodies (circulating antibody 



244 Pesticides in the Modern World - Risks and Benefits 

levels or antibody-forming cell numbers), number of circulating leucocytes, lymphoid organ 
cellularity and weights (Luebke et al., 1997; Bols et al., 2001). 

3.1 Heavy metals 

Heavy metals in aquatic environments are receiving more and more attention. Among the 
adverse effects, they can produce mortality, alteration of sexual maturation or 
immunodeficiency. Some heavy metals may transform into the persistent metallic 
compounds with higher toxicity, which can be bioaccumulated in the organisms and 
magnified in the food chain, thus threatening human health (Zhou et al., 2008). 
Chromium (Cr) is a naturally occurring element found in rocks, animals, plants, and soil, 
predominantly in its insoluble trivalent form [Cr(III)]. Unfortunately, excessive 
industrialization and other anthropogenic activities have led to the global occurrence of 
soluble Cr (VI) in concentrations above permissible levels (Velma et al., 2009). The very 
scarce data in vitro have demonstrated that incubation of common carp (Cyprinus carpio) 
leucocytes with 2-200 |*M hexavalent chromium showed depressed lymphocyte 
proliferation upon mitogen induction, as well as phagocytic functions, at much lower 
concentrations that produced cytotoxicity or cell death (Steinhagen et al., 2004). Moreover, 
neutrophils changed their morphology and reduced the amount of ROS and RNI. In vivo 
studies are more abundant and diverse and have also demonstrated the direct negative 
effects on fish leucocyte function and viability. Thus, tilapia (Oreochromis mossambicus) 
specimens exposed to sublethal doses of Cr-containing tannery effluents suffered a 
decreased antibody production, serum lysozyme activity and production of ROS and RNI 
by peripheral blood leucocytes (Sudhan & Michael, 1995; Prabakaran et al., 2007). Tilapia 
specimens exposed for 28 days with 0.5 and 5 mg Cr (VI)/L also decreased the disease 
resistance to bacterial infection and non-specific and specific immune response whilst the 
exposure with 0.05 mg Cr (VI)/L produced the opposite effects (Prabakaran et al., 2006). In 
another study, the spleen weight and the lymphocyte and leucocyte counts were 
significantly reduced by chronic exposure to Cr (III) and Cr (VI), producing the hexavalent 
form the greatest inhibitions (Arunkumar et al., 2000). In Tilapia sparrmanii, acute or chronic 
water exposures to potassium dichromate (0.098 mg/L) produced general haematological 
disorders including thrombocytopenia (Gey van Pittius et al., 1992). Moreover, and 
depending on the pH, fish showed leucocytosis and leucopenia at acidic and basic pH 
values, respectively (Wepener et al., 1992). In another more extensive study, the freshwater 
fish Saccobranchus fossilis were exposed for 28 days to 0.1-3.2 mg Cr (IV)/L and showed 
important changes in humoral and cellular immune responses and disease resistance 
(Khangarot et al., 1999). Concretely, they found a significant increase in the spleen size 
accompanied by an increment of spleenic lymphocytes. However, the number of plaque- 
forming cells and the phagocytic activity was reduced in spleen and head-kidney 
leucocytes. On the other hand, at blood level, the number of lymphocytes was decreased, 
but neutrophils and thrombocytes were increased, as well as the level of circulating 
antibodies and resistance to Aeromonas hydrophila infections. Otherwise, in plaice 
(Pleuronectes platessa), Cr-treatment increased the number of melano-macrophage centres but 
reduced their size (Kranz & Gercken, 1987). In the case of common carp and brown trout 
(Salmo trutta L.), 38 weeks of exposure with potassium dichromate diminished the primary 
and secondary humoral responses being the carp more susceptible to the heavy metal 
(O'Neill, 1981). In other kind of studies, the chromium exposure was carried out by dietary 



Immunotoxicological Effects of Environmental 

Contaminants in Teleost Fish Reared for Aquaculture 245 

intake and resembling the food chain bioaccumulation. In this case, rainbow trout 
(Oncorhynchus mykiss) fed diets containing 1540 to 4110 ppb Cr showed increased serum 
lysozyme activity as well as respiratory burst and phagocytic activity of macrophages in a 
dose- and time-dependent manner (Gatta et al., 2001). 

Mercury (Hg), and derivatives such as methylmercury, are also important contaminants in 
aquatic environments inducing organ lesions, neurological, haematological and 
immunological disorders (Sweet & Zelikoff, 2001). First evidences, in rainbow trout, 
described a decrease in the number of mucous-producing cells and mucus production after 
exposure to mercury and methylmercury, which can be associated to impaired immunity 
(Lock & Overbeek, 1981). Afterwards, serum C-reactive protein was increased in freshwater 
murrel {Ghana punctatus) (Ghosh & Bhaattacharya, 1992) and major carp (Catla catla) (Paul et 
al., 1998) by exposure to mercury. However, plasmatic lysozyme of plaice was decreased 
after exposure to sublethal doses of mercury (Fletcher, 1986). In sharp contrast, blue 
gourami (Trichogaster trichopterus) showed increased kidney and plasma lysozyme activity, 
but at the same time reduced the production of agglutinating specific antibodies after 
chronic exposure to 0.045 or 0.09 mg Hg2+/L (Low & Sin, 1998). Further evidences have 
been obtained in vitro. Blue gourami lymphocytes incubated with mercury showed 
increased proliferation at low dosages, which was reversed by higher levels (>0.045 mg/L) 
(Low & Sin, 1998). In the marine fish Sciaenops ocellatus, mercury treatment (<10 |aM) 
produced a high-dose inhibition and a low-dose activation of leukocytes as determined by 
Ca-mobilization and tyrosyne phosphorilation of proteins (MacDougal et al., 1996). More 
recently, in the European sea bass (Dicentracrchus labrax), in vitro treatment with HgCk 
induced apoptosis in head-kidney macrophages as well as reduced the ROS production and 
the benefits of macrophage-activating factors (MAF) (Sarmento et al., 2004). 
Cadmium (Cd) is a nonessential heavy metal causing great toxicity. Among the first 
observations, Robohm (1986) found that Cd treatment inhibited the antibody levels in 
cunners (Tautogolabrus adspersus) and enhanced the antibody levels and chemotactic activity 
of peritoneal exudate cells in striped bass (Morone saxatilis). In rainbow trout exposed to 2 
ppb of Cd, the same level found in some contaminated waters, the lysozyme activity was 
unaffected while the macrophage functions, phagocytosis and production of ROS, were 
significantly impaired (Zelikoff et al., 1995). These authors also demonstrated that Japanese 
medaka (Oryzias latipes) leucocytes increased their production of ROS and phagocytic 
functions without any change in many haematological parameters or antibody levels 
(Zelikoff et al., 1996). In the European sea bass, while in vivo exposure had a similar 
inhibitory effect on phagocytic functions the in vitro treatment produced an increment 
(Bennani et al., 1996). In the case of juvenile common carp experimentally infected with the 
blood parasite, Sanguinicola inermis (Trematoda: Sanguinicolidae) there were tissue changes 
and while the counts of neutrophils, eosinophils and thrombocytes increased in the thymus 
the number of neutrophils in the pronephros was reduced due to Cd2+ treatment (0.1 mg/L) 
(Schuwerack et al., 2003). More recently, the Cd exposure has been related to the increase of 
melano-macrophage centres on several fish tissues (Suresh, 2009). In the hybrid tilapia 
(Oreochromis niloticus x O. aureus), the Cd exposure increased the lysozyme activity but 
greatly reduced the alternative complement activity (Wu et al., 2007). 

Copper (Cu) is an essential nutrient but intensive use against fungal infections has shown to 
become a contaminant in some aquatic environments with immunosuppressive effects in 
general. S.fossilis fish exposed to sublethal Cu concentrations (0.056 to 0.32 mg/L) adversely 



246 Pesticides in the Modern World - Risks and Benefits 

affected the humoral and cell-mediated immune system parameters (Khangarot et al. 1988; 
Khangarot & Tripathi, 1991) and reduced the fish resistance to A. hydrophila infections 
(Khangarot et al., 1999). European sea bass exposed to copper also showed an inhibited 
phagocytosis and ROS production both in vivo and in vitro (Bennani et al., 1996). Similar 
findings were also recorded in other experimental fish such as rainbow trout, goldfish 
(Carassius auratus), Puntius gonionotus or Colossoma macropomum (Hetrick et al. 1979; Knittel, 
1981; Muhvich et al., 1995; Shariff et al, 2001; Lugo et al, 2006). Both in vitro and in vivo data 
have also demonstrated a decrease in the NCC activity and phagocytic responses in 
zebrafish (Danio rerio) (Rougier et al., 1994). Strikingly, further studies in common carp have 
shown increased humoral and cellular immune responses after Cu treatment (0.1-2.5 mg/L) 
(Dautremepuits et al., 2004a, 2004b). Very recently, Cu-incubation of trout macrophages up- 
regulated the expression of immune-relevant genes (interleukin-l(3 (IL-1J3), IL-6, tumor 
necrosis factor-a (TNFa), serum amyloid A (SAA) and trout C-polysaccharide binding 
protein (TCPBP)) trying to understand the mechanisms and regulation of the immune 
response by heavy metals (Teles et al., 2011). 

The immunotoxic impact of other heavy metals in fish has received less attention. Thus, zinc 
(Zn) was able to induce lymphoproliferation and NK-cell activity against tumor cells in 
common carp pronephros (Ghanmi et al., 1989, 1990). In zebrafish kidney leucocytes, Zn 
treatment increased the NCC activity and reduced the phagocytic responses both in vitro 
and in vivo (Rougier et al., 1994). MnCk treatment also increased lymphoproliferation and 
NK cell activity in carp (Ghanmi et al., 1989, 1990). By contrast, Ni exposure reduced the 
lymphoproliferative response in medaka and deeper analysis led to the authors to suggest 
that the targets were the T-cells since neither the LPS-induced B-cell proliferation and 
antibody-forming cells were unaffected (Luebke et al., 1997). Arsenic (As) reduced the 
leucocyte respiratory burst, expression of some immune-relevant genes and disease 
resistance in zebrafish (Hermann & Kim, 2005; Nayak et al., 2007) in a similar fashion than 
in the catfish Clarias batrachus (Ghosh et al., 2007; Datta et al., 2009). 

3.2 Polycyclic aromatic hydrocarbons (PAHs) 

Aquatic environments are usually contaminated by PAHs derived form industry or 
petroleum, which produce external abnormalities, somatic mutations, cancer and 
immuno depression (Skupinska et al., 2004). The most toxic and the best studied are 7,12- 
dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BaP) and 3-methylcholanthrene (3- 
MC) (Davila et al., 1995). In fish, as in mammals, the immunotoxicological effects are 
somehow contradictory and depend on the dose and time of exposition. 
Liquid creosote (3-10 (il/L), containing PAHs, exposure of rainbow trout produced 
decreased respiratory burst of head-kidney leucocytes but increased phagocytic activity and 
percentage of Ig+ cells at short exposition times (Karrow et al., 2001). However, after 28 
days, respiratory burst and phagocytic activity returned to control levels while the count of 
B cells remained decreased. The use of the heavily polluted Elizabeth River (Virginia, USA) 
has been extensively used for immunotoxicological evaluations. In the case of mummichogs 
(Fundulus heteroclitus), contamination produced a decrease in the levels of circulating IgM, 
both total and specific, and NCC activity while the plasmatic lysozyme was increased 
(Faisal et al., 1991a; Frederick et al., 2007). Moreover, lymphoid cells expressed higher levels 
of lysozyme and COX-2 (cyclooxygenase-2), the last as indicator of macrophage activation. 
Native fish (Leiostomus xanthurus and Trinectes maculates) from this river also showed lower 



Immunotoxicological Effects of Environmental 

Contaminants in Teleost Fish Reared for Aquaculture 247 

chemotactic and phagocytic activities that those kept in clean waters, and this suppression 
was reversed by maintenance in clean waters for several weeks (Weeks & Warinner, 1984; 
Weeks et al., 1986). Treatment of rainbow trout with 10-70% sewage plant effluents 
(containing PAHs among other contaminants) also reduced the number of circulating 
lymphocytes but increased their in vitro proliferation capacity (Hoeger et al., 2004). 
Strikingly, this effluent failed to alter any other immune functions such as respiratory burst, 
phagocytosis, lysozyme activity, leucocyte populations other than lymphocytes and A. 
salmonicida-speciiic IgM production. Intraperitoneal (ip) injection of diesel oil-based drilling 
mud extracts produced no effect on IgM levels and complement activity, suppression of 
serum lysozyme and elevated head-kidney lymphocyte proliferation in response to 
phytohemagglutinin (Tahir & Secombes, 1995). Petroleum-containing sediments also 
affected the immune response of flounder (Pseudopleuronectes americanus) since the number 
of melano-macrophage centres were diminished (Payne & Fancey, 1989). Deeper studies 
have evaluated the effects of heavy oil contamination (3.8 g/L for 3 days) in the Japanese 
flounder (Paralichthys olivaceus) using cDNA microarrays (Nakayama et al., 2008). They have 
found an alteration of expression in immune-related genes including down-regulation of 
immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and 
macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and 
lysozyme. Moreover, in vitro incubation with oils, pure and single PAHs, of European sea 
bass plasma produced significant changes in lysozyme and alternative complement 
activities indicating that these contaminants caused changes in the production of them by 
the leucocytes but also directly affects the enzymatic activity (Bado-Nilles et al., 2009). 
Similarly, PAHs mixture spiked-sediments (10 mg/kg dry wt) failed to change the serum 
lysozyme but reduced the ROS activity of kidney leucocytes of dab (Limanda limanda) 
(Hutchinson et al., 2003) while decreased the number of circulating lymphocytes (Khan, 
2003). In the marine fish spot, L. xanthurus, exposed to PAH-contaminated sediments the T- 
lymphocyte proliferation was suppressed but the B-cell proliferation was greatly increased 
(Faisal et al., 1991b). Rainbow trout fed diets containing 0.66 or 7.82 |a.g PAH mixtures/g 
bw/day resulted in suppressed disease resistance against bacteria (Bravo et al., 2011). 
Regarding the effects of single and pure PAHs, injections of DMBA (0.6 or 12.7 mg/kg body 
weight-bw) depressed the number of plaque-forming cells in head-kidney and spleen to T- 
independent antigens in Chinook salmon (Oncorhynchus tshawytscha) (Arkoosh et al., 1994). 
Injection of tilapia (Oreochromis niloticus) with DMBA (25 or 75 mg/kg bw) produced 
hipocellularity in spleen and head-kidney whilst phagocytosis and respiratory burst activity 
were not altered unless mortality occurred (Hart et al., 1998) similarly to the unaffected trout 
phagocytosis (Spitsbergen et al., 1986). By contrast, i.p. injection of 1-100 mg DMBA/kg bw 
to oyster toadfish (Opsanus tau) resulted in a peritoneal macrophage activity suppression in 
essentially a linear fashion, whereas NCC activity was virtually obliterated at all dosages 
(Seeley & Weeks-Perkins, 1997). BaP suppressed B cell immunity in tilapia at 15 mg/kg 
while increased at 25 mg/kg (Smith et al., 1999). Injection of 5-50 mg/kg also produced 
important histological changes in pronephros (reduction of lymphoid elements and 
augmentation of immune cells in apoptosis) and while the phagocytic activity was unaltered 
the respiratory burst was reduced (Holladay et al, 1998). In Japanese medaka, BaP injection 
(2-200 mg/kg bw) greatly reduced lymphocyte proliferation and number of antibody- 
forming cells (Carlson et al., 2002, 2004). In European sea bass, ip injection of BaP (20 mg/kg 
bw) significantly depressed the leucocyte phagocytosis and completely abrogated the ROS 



248 Pesticides in the Modern World - Risks and Benefits 

production (Lemaire-Gony et al., 1995). In rainbow trout, BaP and BaA (benzo(a)anthracene) 
injection failed to significantly change the phagocytic activity (Walczak et al v 1987). Finally, 
3-MC injection (40 mg/kg bw) into common carp increased the proliferative ability of 
resting circulating lymphocytes, rainbow but reduced their proliferative activity with the B- 
and T- mitogens, as well as the macrophage respiratory burst (Reynaud et al., 2002, 2003; 
Reynaud & Deschaux, 2005). Similarly, trout exposed to 3-MC increased the serum C- 
reactive protein 10-20-fold but not affected the IFN activity of leucocytes, measured as the 
resistance to bluetongue virus (Winkelhake et al., 1983). 

3.3 Organochlorinated (OCs) contaminants 

This group of contaminants comprises many of the most toxic and persistent compounds for 
aquatic environments such as DDT and relatives, lindane, polychlorinated biphenyls (PCBs), 
polychlorinated dibenzo-p-dioxins (PCDDs or dioxins) or polychlorinated dibenzofurans 
(PCDFs or furans). These are common contaminants in water ecosystems and their residues 
still have toxic consequences including immunotoxicity, reproductive deficits, 
teratogenicity, endocrine toxicity and carcinogenicity (Ahlborg et al., 1994). Unfortunately, 
although OC levels detected in fish worldwide seems to be declining they still should be 
lowered to decrease risk for human consumers (Gomara et al., 2005). 

DDT (l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane), and its metabolites DDE (p,p'-DDE and 
o,p-DDE), are among the most important OCs in agricultural and aquatic environments. 
However, though no information exists regarding the direct effect of DTT on fish 
immunology some data are available about its derivatives. Thus, o,p-DDE treatment (10 
ppm) of Chinook salmon, at fertilisation and hatch stages, failed to affect viability and 
growth but these fish still suffered immunosuppression one year later as consequence of the 
contamination (Milston et al., 2003). In vitro, p,p'-DDE (0-15 mg/L) produced a reduction in 
lymphocyte-granulocyte viability, by increasing the percentage of apoptotic cells, and in 
lymphocyte proliferation, in both spleen and head-kidney that was also observed in vivo (59 
ppm exposure) (Misumi et al., 2005). By contrast, marine gilthead seabream leucocytes 
incubated with p,p'-DDE (5 ng to 50 mg/ml) failed to change their viability and main innate 
cellular immune parameters but up-regulated the expression of some immune genes (IL- 
lbeta, TNFalpha, MHCIalpha, MHCIIalpha, Mx, TLR9, IgM and TCRalpha) indicating only 
effects at genetic level but not in function (Cuesta et al., 2008b). 

Lindane (gamma-hexachlorocyclohexane) is another OC that have focused much of the 
attention. Dietary intake of lindane (10-1000 ppm) failed to affect the spleen weight, serum 
and mucus antibody levels and phagocytosis in the common carp though most of the tissues 
reflected great contamination (Cossarini-Dunier, 1987; Cossarini-Dunier et al., 1987). In 
rainbow trout, intraperitoneal injection of lindane (10-100 mg/kg bw) greatly depressed the 
number of antibody-secreting cells, serum lysozyme levels, respiratory burst activity and 
myeloperoxidase (contributes together with ROS and RNI to pathogen killing), proliferating 
capacity of B cells, but not of T cells, and its percentage in the head-kidney but at the same 
time increased the plasmatic ceruloplasmin, an acute phase protein (Dunier & Siwicki, 1994; 
Dunier et al., 1994). The same group also demonstrated that oral administration of lindane (1 
mg/kg) for 30 days significantly decreased the respiratory burst activity of head-kidney 
leucocytes but unaffected the lymphocyte proliferation and number of circulating B 
lymphocytes in a similar way to the previous data in carp (Cossarini-Dunier et al., 1987; 
Dunier et al., 1994). Moreover, they have also demonstrated that these negative effects can 
be reversed by the in vitro addition of nitrogranulogen (Siwicki & Dunier, 1994) or dietary 



Immunotoxicological Effects of Environmental 

Contaminants in Teleost Fish Reared for Aquaculture 249 

intake of vitamin C (Dunier et al v 1995). Lindane bath of Nile tilapia also reduced the counts 
of circulating leucocytes, phagocytic activity and antibody levels (Khalaf-Allah, 1999). In 
vitro, lindane (2.5-100 jiM) treatment was able to increase ROS production in rainbow trout 
head-kidney phagocytes and MAF (macrophage activating factors) production by 
peripheral blood leucocytes, in both cases depending on the dose and with contradictory 
results (Betoulle et al., 2000; Duchiron et al., 2002a, 2002b). These studies also demonstrated 
that low lindane concentrations increase the cytoplasmatic cAMP but high doses increase 
the intracellular Ca2+, and these two factors contribute to the dual effects of 
induction/ reduction of the leucocyte immune functions produced by lindane treatment in 
leucocytes (Betoulle et al., 2000; Duchiron et al., 2002a, 2002b). In gilthead seabream, head- 
kidney leucocyte incubation (5 ng to 50 jig/ ml) with lindane failed to significantly change 
the leucocyte viability (by necrosis and apoptosis) and innate cellular immune functions 
(phagocytosis, respiratory burst and cell-mediated cytotoxicity) but strikingly increased the 
expression of many immune-related genes (IL-lbeta, TNFalpha, MHCIalpha, MHCIIalpha, 
Mx, TLR9, IgML and TCRalpha) (Cuesta et al., 2008b). 

PCBs, with theoretically 209 distinct congeners, may be divided into those with coplanar 
geometry, the most toxic as they bind and activate AhR (hydrocarbon receptors) and CYP1A 
(cytochrome P4501A) expression, while noncoplanar congeners can interfere with AhR 
signalling but also affect cells via AhR-independent pathways (Duffy & Zelikoff, 2006). 
Immunotoxicological effects of PCB mixtures, such as Arochlor, have been evaluated in fish. 
Thus, Aroclor 1254 depressed plaque-forming cells in head-kidney and spleen to a T- 
independent antigen in Chinook salmon after ip injection (Arkoosh et al., 1994). However, it 
failed to modulate the innate disease resistance and antibody production by oral 
administration of environmental doses in the same fish (Powell et al., 2003). In Artie charr 
(Salvelinus alpinus), diets containing 100 mg Aroclor 1254/ kg diet resulted in increased disease 
susceptibility to furunculosis (Maule et al., 2005). In Atlantic salmon (Salmo salar), by contrast, 
water exposure with 1-10 |ig/L produced increased T lymphocyte proliferation at short and 
long-term (Iwanowicz et al., 2005). In rainbow trout, while the C-reactive protein levels in 
serum were increased the leucocyte IFN and NCC activities were unchanged (Winkelhake et 
al., 1983; Cleland & Sonstegard, 1987). Another study using Aroclor 1248, in the brown 
bullhead (Ameiurus nebulosus), have provoked a decrease in the bactericidal activity and 
antibody titers (Iwanowicz et al, 2009). PCBs mixture (Aroclor 1242, 1254 and 1260) failed to 
modify lysozyme and ROS activity in L. limanda (Hutchinson et al., 2003). Regarding the effects 
of pure PCBs, the congener 126 has been the most studied. PCB 126 injection (0.01-1 fig/g bw) 
to Japanese medaka reduced the antibody forming cell numbers (Duffy et al., 2002) but either 
reduced or increased the phagocyte-mediated ROS production at 3 or 14 days post-treatment, 
respectively (Duffy et al., 2003). Dietary administration (100 ng/g bw) to European eel 
(Anguilla anguilla) completely abrogated the production of specific antibodies against a 
parasite (Sures & Knopf, 2004). PCB 126 also produced a reduction of phagocyte respiratory 
burst and NCC activities in channel catfish (Ictalurus punctatus) at (0.01-1 mg/kg bw) (Rice & 
Schlenk, 1995). In the bluegill sunfish (Lepomis macrochirus), the coplanar PCB 126 (0.01 or 1.0 
ug/g bw) also slightly affected the B-lymphocyte proliferation while the noncoplanar PCB 153 
(5.0 or 50.0 ug/g bw) significantly reduced the phagocyte-mediated respiratory burst activity 
and the B- and T- lymphocyte proliferation (Duffy & Zelikoff, 2006). Strikingly, short 
incubation of rainbow trout head-kidney leucocytes with PCB 126 (1 \iM) increased the 
expression of IL-lp gene and failed to abrogate the LPS effects on gene regulation (Quabius et 
al, 2005). The PCB Clophen A50 (0.4-2 jig/egg) injected into the eggs of rainbow trout with 



250 Pesticides in the Modern World - Risks and Benefits 

pathogenic bacteria resulted in a higher disease resistance than those injected with the bacteria 
suggesting a direct effect on the immune response (Ekman et al., 2004). 

Chlorinated dioxins, as typified by the most potent isomer TCDD (2,3,7,8- 
tetrachlorodibenzo-p-dioxin), are also very toxic for fish. Injection of 0.1-10 ng TCDD/kg bw 
to rainbow trout resulted in very little changes in humoral and cellular immune responses 
(Spitsbergen et al., 1986). However, while the C-reactive protein levels in serum were 
increased the leucocyte production of IFN was unchanged (Winkelhake et al., 1983). In 
common carp, TCDD injection produced histological alterations including increase of 
melano-macrophage centres and reduction of lymphocyte numbers (van der Weiden et al., 
1994). Further studies have also evaluated fish tissue alterations and CYP1A staining 
patterns have been described in European flounder (Platichthys flesus) and gilthead 
seabream (Grinwis et al., 2000; Ortiz-Delgado & Sarasquete, 2004). 

Some studies have also evaluated the immunotoxicological effect of other OCs. In the case of 
furans (PCDF), most authors have focused on other fish toxicity tests rather than in 
immunotoxicology. Endosulfan exposure produces developmental and neurological 
disorders and acts as endocrine disruptor. Rainbow trout leucocyte treatment with 
endosulfan inhibited the lymphoproliferative activity where the B-cells were more sensitive 
than the T lymphocytes (O'Halloran et al., 1996). In another study, crimson-spotted 
rainbowfish (Melanotaenia fluviatilis), golden perch (Macquaria ambigua) and Murray cod 
(Maccullochella peelii), but not silver perch (Bidyanus bidyanus), leucocytes showed decreased 
phagocytosis after endosulfan treatment (10 mg/L) (Harford et al., 2005). In vivo treatment 
of Nile tilapia for 96 h at 7 ppb produced an increased phagocytosis and ROS production by 
spleen leucocytes, IgM levels and production of IL-2-like, but at the same time reduced the 
spleen viability and relative weight (Tellez-Banuelos et al., 2009, 2010). 

3.4 Organophosphorous pesticides (OPs) 

OPs are insecticides used world-wide as an alternative to the persistent and more 
bioaccumulative OCs. They are potent neurotoxic and immunotoxic since are irreversible 
acetylcholinesterase inhibitors (Galloway and Handy, 2003). Malathion exposure (0.2-0.8 
mg/L) of medaka resulted in reduced number of antibody-forming cells but unchanged 
circulating leucocyte numbers and T-cell proliferation (Beaman et al., 1999). Vaccinated Nile 
tilapia exposed to malathion or diazinon presented lower blood cell counts, phagocytosis 
and antibody levels than those unexposed (Khalaf-Allah, 1999). Diazinon exposure of 
bluegill had biphasic effects with immune response increases at low concentrations and 
depressions at high dosages (Dutta et al., 1997). In Nile tilapia, Giron-Perez et al., (2007, 
2008, 2009) have showed that diazinon altered the spleen counts and lymphocyte 
proliferation, serum IgM and lysozyme levels, phagocytic activity and respiratory burst 
depending on the exposure dose and time. Chlorpyrifos displayed little immunotoxicity, 
although there was a dose-dependent reduction in Murray cod lymphocytes (Harford et al., 
2005). Nile tilapia exposed to the LC50 failed to change blood parameters but the phagocytic 
activity was significantly reduced (Giron-Perez et al., 2006). Chlorpyrifos exposure 
produced an up-regulation of hsp60, hsp70 and hsp90 genes, related to the cellular stress 
response in Chinook salmon. Moreover, the cytokine (IL-lb, TGF-beta, Mx and insulin 
growth factor (IGF)-I) gene expression was unaltered or down-regulated but not affected the 
virus susceptibility of the fish (Eder et al., 2008, 2009). Dichlorvos and trichlorfon 
insecticides have been used in aquaculture against ectoparasites in the past. Trichlorfon 
exposure decreased the serum lysozyme, lymphocyte proliferation, respiratory burst and 



Immunotoxicological Effects of Environmental 

Contaminants in Teleost Fish Reared for Aquaculture 251 

phagocytosis of common carp leucocytes (Siwicki et al., 1990; Dunier et al., 1991) but 
unchanged the production of specific antibodies (Cossarini-Dunier et al., 1990). Water 
exposure to dichlorvos failed to change the specific IgM production but altered other serum 
innate immune parameters (Dunier et al., 1991). Edifenphos and glyphosate exposure 
reduced the lymphocyte proliferation, antibody-producing cells and circulating IgM levels 
in Nile tilapia (el-Gendy et al, 1998). Glyphosate exposure of silver catfish (Rhamdia quelen) 
resulted in decreased phagocytosis and resistance to disease (Kreutz et al., 2010). 

3.5 Pyrethorids 

Pyrethroids are extensively used insecticides since they are very stable and produce low 
mammalian toxicity but this is very high for aquatic animals (Bradbury & Coats, 1989). 
Among them, deltamethrin injection to Ancistrus multispinis increased peritoneal leucocyte 
numbers and production of RNI by macrophages (Pimpao et al., 2008). Short exposure to 
deltamethrin (30 min., 1-4 ng/L) of rainbow trout resulted in decreased serum lysozyme 
and IgM levels (Siwicki et al., 2010). Water exposure of rohu (Labeo rohita) to alpha- 
permethrin produced a reduction in lysozyme activity and resistance to bacteria (Nayak et 
al., 2004). Rainbow trout exposure to cypermethrin failed to alter any of the immune 
parameters (Shelley et al., 2009). Esfenvalerate exposure produced an up-regulation of 
hsp60, hsp70 and hsp90 stress genes, down- or non-regulated cytokines and unaffected the 
virus susceptibility of the Chinook salmon (Eder et al, 2008, 2009). Using microarrays, delta 
smelt (Hypomesus transpacificus) exposure to esfenvalerate produced alterations in the 
expression of genes associated with immune responses, along with apoptosis, redox, 
osmotic stress, detoxification, growth and development (Connon et al., 2009). 

3.6 Organotins 

Organotin compounds or stannanes are chemical compounds based on tin (Sn) with 
hydrocarbon substituents showing different toxic effects. TBT (triorganotins) is specially 
important since it has been widely used as marine anti-biofouling agent. Injection of 0.01-1 
mg TBT (tributyltin)/kg bw of channel catfish altered leucocyte counts, NCC, phagocytic 
and respiratory burst activities, production of specifc antibodies and number of antibody- 
produceing cells (Rice et al., 1995). TBT treatment signinficantly reduced the lymphocyte 
numbers in spleen, the thymus volume and the leucocyte NCC activity in European 
flounder (P. flesus) (Grinwis et al., 2000). In rainbow trout, in vitro incubation with 2.5-500 
ppb TBT and DBT (dibutyltin) reduced the lymphoproliferation activity in pronephros and 
spleen but failed to affect the NCC activity showing DBT higher toxicity than TBT 
(O'Halloran et al., 1998). In vitro incubation of several Australian fish head-kidney 
leucocytes with TBT or DBT depressed the phagocytic activity and reduced the numbers of 
lymphocytes and granulocytes (Harford et al., 2005). 

3.7 Other chemicals 

Herbicides are still widely used and end in aquatic environments producing many 
physiological alterations but little studies have focused on their immunotoxicological effects 
in fish. Herbicides mixture, containing atrazine, simazine, diuron and isoproturon, 
exposition of goldfish increased spleen and head-kidney ROS production and serum 
lysozyme but reduced the specific antibodies and resistance to bacterial infections (Fatima et 
al., 2007). Atrazine exposure of silver catfish resulted in decreased phagocytosis and 



252 Pesticides in the Modern World - Risks and Benefits 

resistance to disease (Kreutz et al v 2010) whilst failed to do so in common carp (Cossarini- 
Dunier et al., 1987; Cossarini-Dunier & Hattenberger, 1988). Phenols are another group of 
toxics. Phenol, pyrocatechol and hydroquinone decreased the cell-mediated cytotoxic 
activity of spleen lymphocytes in common carp (Taysse et al., 1995), pentachlorophenol 
reduced macrophage production of cytokines in goldfish (Chen et al., 2005) but activated 
phagocytosis and unaltered other immune functions and disease resistance in rainbow trout 
(Shelley et al., 2009). Endocrine disrupting chemicals produce population decline, an 
increasing incidence of cancer, inhibition of reproductive function, and developing 
disruption of the immune and nervous systems. However, there are very limited data 
concerning the role of endocrine disrupting chemicals on aquatic organism, including the 
fish immune response. Zebrafish embryos exposed for 3 days to 17a-ethynyestradiol, 
permethrin, atrazine and nonylphenol (0.1-12.5 ug/L) altered the expression of immune- 
relevant genes (TNFa, IFN, IL-lp\ IL-8, CXCL-Clc, CC-chemokines, iNOS, etc.) indicating 
their single and combined effects upon fish immune response (Jin et al., 2010). 

4. Conclusion 

As described above, most of the aquatic contaminants have shown either activations or 
suppressions in the immune response that greatly varied with the exposure route, time, 
dosage and fish specie with many similarities to immunotoxicological data in mammals. 
Therefore, although researchers do not have precise contamination biomarkers in aquatic 
animals some conclusions may rise: i) heavy metals contamination is usually followed by 
metallothionein overexpression (Misra et al., 1989; Hansen et al., 2007; Costa et al., 2009); ii) 
OCs exposure is concomitant to decreased number and size of melano-macrophage centres 
(Schmitt et al., 2005; Hinck et al., 2007); iii) immunotoxicological effects due to PHAs and PCBs 
are generally parallel to an increase in the activity of the detoxification proteins cytochrome 
P4501A (CYP1A), through the involvement of aryl hydrocarbon receptors (AhR), and/or 
EROD (ethoxyresorufin-O-deethylase) (Lee & Anderson, 2005; Duffy & Zelikoff, 2006; 
Reynaud & Deschaux, 2006; Bravo et al., 2011); and iv) further and deeper studies are needed 
to understand the real effect of environmental contaminants in fish and the mechanisms for 
toxicity. Moreover, looking at the fish species studied and those subjected to aquaculture, most 
of the data come from wild fish, salmonids and cyprinids but other major species are almost 
ignored. Even further, most of the studies focus on freshwater fish and very little is known for 
marine species. These aspects should be covered by future works to progress in the 
understanding of the immunotoxicological effects and mechanisms and the consequences and 
risks they may have on human consumers as consequence of the bioaccumulation. 

5. Acknowledgments 

This work has been funded by national (IDI-20091041, AGL2008-05119-C02-01 and 
AGL2010-20801-C02-02) and regional projects (04538/GERM/06). A. Cuesta thanks to the 
Ministerio de Ciencia e Innovation for the Ramon y Cajal contract. 

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14 



Using Zooplankton, Moina Micrura Kurz to 

Evaluate the Ecotoxicology of Pesticides 

Used in Paddy Fields of Thailand 

Chuleemas Boonthai Iwai 1 , Atcharaporn Somparn 1 and Barry Noller 2 

department of Plant Science and Agriculture Resource, 

Land Resources and Environment Section, Faculty of Agriculture, Khon Kaen University, 

2 The University of Queensland, Centre for Mined Land Rehabilitation (CMLR), 

1 Thailand 
2 Australia 

1. Introduction 

Thailand is an agricultural country where agriculture is a very important part of the economy. 
Thailand expanded exports of agricultural products and also imports fertilizers and pesticides 
intensively. Pesticides are used widely in agriculture and trade of agricultural products to 
increase agricultural yield and to protect plant from diseases, weeds and insect damage 
(Department of Agricultural, 2010). Since pesticides were first imported into Thailand under 
the "Green Revolution Policy" as part of the 1st National Economic and Social Development 
Plan in 1966, the total amount of imported pesticides has dramatically increased year by year. 
Most pesticides used in the country are imported (Department of Pollution Control, 2005), and 
the quantities of imported agricultural pesticides have increased 3 times from 1994 to 2005, 
reaching more than 80 thousand tonnes in 2004. Pesticides are applied in the highest quantity 
in vegetable and fruit farming, where market pressure for appearance is higher. In 2000, 
organophosphates contributed the majority of imported pesticides followed by carbonates and 
organochlorines; most were herbicides, followed by insecticides, disease control agents and 
plant growth regulators (Department of Pollution Control, 2002). 

The result from increasing pesticides uses has resulted in significant increased crop 
contamination and human health hazard (Office of Epidemiological, 2009). The risk of 
pesticide contamination in fruits and vegetables in Thai market often occurs. 
Rice is the major crop and food source for most Asian countries including Thailand. Rice 
production from paddy fields faces variety of pests that require a range of pesticides and 
herbicides to manage the presence of insects and weeds, as well as fungal and bacterial 
pathogens. Indeed, losses of the total world rice crop due to insects have been estimated to 
occur at a rate of 28% per annum, which is four times greater than the average for other 
world cereal crops. More than 90% of the global end-user market in pesticides for rice 
production is applied in Asia (Abdullah, 1995). In Thailand, pesticides play an important 
part and widely use on rice production because its benefits in pest control and increased rice 
production. Therefore, pesticide contamination in draining water from paddy field has been 
one of non-point source pollution in aquatic ecosystem (Sanchez et al, 2006). 



268 Pesticides in the Modern World - Risks and Benefits 

This is attributed to be relatively large amounts pesticides applied in paddy field, in addition 
to common practice of draining the paddy water in draining canals (Tejada, 1995). Around 95 
% of freshwater in Thailand is withdrawn to irrigate the more than 5 million hectares of 
irrigated agriculture. Waste water from this activity may pose significant environmental 
hazards for aquatic ecosystem in particularly aquatic biota. Furthermore, this contaminate 
affect wildlife species ether by direct exposure or through bioaccumulation in food web. 
Pesticide contamination sites associated with paddy field activities may pose significant 
environmental hazards for terrestrial and aquatic ecosystems. They are important sources of 
agro-sourced pollution and may result in ecotoxicological effects, particularly following 
transfer of irrigation waters following use. Ecotoxicological effects occur at all trophic levels, 
from the molecular to the ecosystem level and effects may be observed via biomonitoring 
with both individual organisms and the ecosystem function and structure. 
Pesticide monitoring is traditionally based on evaluations of individual pesticides identified 
through chemical analyses. A variety of techniques may permit an examination of actual 
pesticides, herbicides and their metabolites that are present (Iwai et al., 2007). These 
techniques are based on sampling approaches that use concentration following collection or 
during collection. Although these techniques still are not able to show the direct response 
that ecotoxicify gives, they do give an indication of what is inducing the response of the 
organism. However, chemical analyses obviously do not reveal complex interaction 
phenomena and polar degradation products are often missed. In contrast to the use of 
chemical analyses, the ecotoxicify bioassay approach integrates the biological effects of all 
compounds present and factors such as bioavailability, synergism, or antagonism are 
reflected directly in the bioassay results. 

Ecotoxicological assessment of pesticides in paddy field are therefore expected to give a more 
comprehensive indication of environmental effects. The use of ecotoxicological assessment to 
evaluate the impact of pesticide residues in the paddy field is strongly recommended in order 
to have a more direct and integrated estimate of environmental impact. In fact, biological 
response to a complex mixture of chemicals integrates different factors such as pH and 
solubility, antagonism or synergism, and the bioavailability of substances. 
Pesticides contamination associated with paddy field has been increased a big concern in 
Thailand. For risk assessment study on the impact of pesticides on aquatic environments 
that surrounding area, information about effect of pesticides on local species were limited, 
especially the ecotoxicological data on aquatic organism in Thailand, and it unknown, 
whether ecological effects test guideline developed elsewhere in the world (US. EPA, ATSM 
etc) may be use in Thailand. Countries located in the tropical zone rely, mostly, on data from 
temperate countries about ecotoxicify data. However, this data may be not suitable for 
tropical countries. Due to the difference organisms species, temperature, rainfall, and 
agriculture practices that might greatly influence pesticides behavior (Abdullah et al., 1997) 
and toxicity of pesticides on organisms. Considering the climate adaption of tropical 
species, assessment of effects of pesticide use on local ecosystem should be performed with 
local species since their sensitive to toxicants may differ considerably form temperate 
organism (Domingues et al., 2007). Differential response of organism representing diverse 
physiological capabilities and niches in aquatic system can help focus field studies where 
nontarget effect due to off - site movement of pesticides are suspected. 
Therefore, Thailand need ecological effects test guideline, this guideline typically derived data 
on toxicological response of local organism to environmental contaminant. The toxicity test is 
procedure that involves the exposure of organism to complex environmental sample under 
controled condition to determine if adverse effects have occurred (Edmondson, 1959). 



Using Zooplankton, Moina Micrura Kurz Evaluated 

Ecotoxicology of Pesticides Used in Paddy Field in Thailand 269 

The objective of this study selected the fresh water cladoceran Moina micrura Kurz order 
Cladocera, family Moinidea. In Thailand, this zooplankton is very common in pond, muddy 
pool and paddy field and it can be mass culture by some local fish farmer as a high quality 
fish food. M. micrura is an ideal animal for ecological relevance, wide occurrence, short life 
cycle, genetic uniformity, relative ease of culture in the laboratory and more sensitive to 
toxicants (Wang, 1994; Wongrat, 2001). The present study was determine the acute and 
chronic toxicity of pesticides on M. micrura. The result would be useful as an input to 
developing a biomonitoring tool and using local species test for evaluation pesticide 
contamination in Thailand aquatic ecosystem. 

2. Materials and methods 

2.1 Test organism culture 

The Moina micrura obtained from Fisheries Research Institute, Khon Khaen (Khon Khaen, 
Thailand) and have been maintained in cultured under control laboratory conditions in 
Ecotoxicology and Environmental Sciences Laboratory, Faculty of Agriculture, Khon Kaen 
University, Thailand. The culture was incubated at 25± 2 °C with 16:8 h light:dark 
photoperiod. M. Micrura were cultured using moderately hardwater and fed on single- 
celled green alga, Chlorella vulagaris from axenic culture. The medium, used for zooplankton, 
as well as for experiments, was tap water at the Faculty of Agriculture, Khon Kaen 
University, Thailand. Water was filtered by using 0.45jim polymembrane filter. Dissolved 
oxygen concentration was between 5-7 mg/L and pH was 7-8. The culturing period for one 
generation was 2 weeks before testing. 

2.2 Test chemicals 

The pesticides test were selected from the common pesticides used in paddy filed of 
Thailand. Five selected pesticides were malathion 58% w/v (CAS: 121-75-5), chlorpyrifos 
40% w/v (CAS:2921-88-2), carbofuran 3 % GR (CA&1563-66-2), neem extract 40% w/v 
(CAS:1141-17-6) and glyphosate 36% w/v (CAS:1071-83-6) (Table 1.). Stock solutions were 
prepared by dissolving the pesticide directly in distill water immediately before to each 
experiment. Stock solution were added to each of three replication test beakers ( 50 ml total 
volume) to obtain nominal exposure concentration. Rang in nominal aqueous exposure 
concentration of chlorpyrifos,malathaion, glyphosate, carbofuran and neem extract on M. 
micrura, were arranged in geometic series between 0.5 -0.0005, 1-50, 500-2500, 3-15 and 50- 
250)i g/L respectively. 

2.3 Experiment design 
2.3.1 Acute toxicity test 

Preliminary acute toxicity tests were conducted in order to calculate malathion, chlorpyrifos, 
carbofuran, neem extract and gyphosate LC50 data. All experiments were performed 
according to the US.EPA document OPPTS 850.1010 (1996) for determining 48 h LC50 values 
for M. micrura. Three replication of 10 neonates (<24 h) per treatment and control laboratory 
well - wate were used. The neonates were exposed in a 150 ml glass beaker containing 50 ml 
for each test concentration and control were static bioassay under laboratory. Test 
organisms were not fed during the testing period. Observation motality was made at 24 and 



270 



Pesticides in the Modern World - Risks and Benefits 



48 h, and results recorded. For water quality, temperature, pH, conductivity and dissolved 
oxygen were measured according to APHA (1992). 



Pesticide Group of pesticide 



Chemical name and number Structure 
(Chemical Abstract Service) 



Malathion Insecticide 

(organophosphate) 



Chlorpyrifos Insecticide 

(organophosphate) 



Carbofuran Insecticide 
(carbamate) 



Neem extract Insecticide 

(biopesticide) 



Glyphosate Herbicide (amine) 



0,0-dimethyl 

phosphorodithioate of diethyl 
mercapto- succinate ; CAS no. 
121-75-5 

Phosphorodithioic acid, 0,0- 
diethyl O- (3,5,6-trichloro-2- 
pyridyl) ester; CAS no:2921- 
88-2 

3,3-dihydro-2,2-dimethyl-7- 
benzofuranyl 
methylcarbamate CAS no. 
1563-66-2 

Azadirachin; CAS no. 1141- 
17-6 



N - (Phosphonomethyl) 
glycine; CAS no. 1071-83-6 



o 



o s=p-o 



•fv-kJk, 




Source: Extoxnet (1996 ); Chemical Book (2007); Compendium of Pesticide common name (2008 a, 2008 
b, 2008 c) 

Table 1. Chemical formulation of pesticides tested withM. micrura . 

2.3.2 Chronic toxicity test 

Chronic toxicity of pesticides to M. micrura followed the procedure recommend by US.EPA 
document 6004-91/002 (1994). Based on acute toxicity result, M. micrura were exposure to 
control and concentration test malathion concentration of 0.05, 0.25 and 0.50 ug/L, 
chlorpyrifos concentration of 0.00005, 0.00025 and 0.00045 ng/L, carbofuran concentration of 
0.25,1.00 and 2.50 ng/L, neem extract concentration of 15, 40 and 65 ug/L and glyphosate 
concentration of 50, 250 and 325 ng/L. In the chronic tests, three replication of 10 neonates 
<(24 h )per treatment and control laboratory well - water were used. The neonates were 
exposed in a 50 ml glass beaker containing 30 ml for each test concentration and control. 
Test organism were fed with a concentrated suspension of the green algae, Chlorella sp. Test 
solution and food were renewed completely every day. The measurement of water quality 
at the beginning and end of the test on control and treatments. The number of offspring was 
noted each day used to evaluate the effect of pesticide on reproduction of test organism. 



2.4 Statistic analysis 

The values of lethal concentration 24 and 48 h LC50 and 95 % confidence limit were 
caculated by appropriate statistical method intervals by probit analysis.Data from chronic 



Using Zooplankton, Moina Micrura Kurz Evaluated 

Ecotoxicology of Pesticides Used in Paddy Field in Thailand 271 

test were analyzed using ANOVA with SPSS version 12 statistical software to detected 
variation significances (P<0.05 ) between treatment group and control. 

3. Results and discussion 

Acute toxicity 

Table 2. show the estimated 48-h LC50 for pesticides, with were calculated from standard 

toxicity test with M. micrura. 

Malathion.: Our 48-h M. Micrura LC50 of 10.44 ng/L was compareable to the 48-h LC50 to 

other Cladocera species. This results shown 48-h- LC50 M. Micrura were nealy repoted the 5- 

10 u.g/L for M. marcocopa by Wang et al. (1994) and the 8 and 13 |ig/L that reported by 

Khan et al. (1993) and Siefirt (1987) for D. Magna. Differnce in LC50 value were observed wih 

Cerodahnia dubia have been reported between 1.14 -3.35 ng/L(Hernadez et al.,2004; Maul et 

al., 2006; Nelson et al.,1997,1998; Ankley et al., 1991). 

Chlorpyrifos: Of the five pesticides tested in this study, chlorpyrifos was the most toxic to 

M. Micrura. The 48-h LC50 value for M. micrura was 0.08 u.g/L. Other values in literature 

were higher between 0.13-3.7 ng/L using D. ambigue, D. Magnaand and D. Duplex (Caceres 

et al.,2007; Van Wijngaarden et al., 1993; Barata et al.,2004; Kersting et al.,1997; Van der 

Hoeven and Gerrisen, 1997). 

Carbofuran: The 48-h LC50 for M. micrura obtain in this study 6.96 ng/L is compareable to 

the 48-h LC50Of 2.69 jig/L obtain with C. Dubia (Nerberg et al.,1997) for carbofuran in D. 

magna were higher than concentration tested (6.96 ng/L). In comparison, Poirer (1990); DBR 

(2000) and Dopsikova (2003) found an acute 48-h LC50 were 86.1, 38.6 and 18.7 ng/L 

respectively. 

Neem extract: The present study found that the 48-h LC50 for neem extract in M. micrura was 

196.3 ng/L. The acute toxicity data for D.magna with 48-h LC50 were 570- 1,250 |ig/L (John , 

2001; Stark ,2001; Scott and Kaushik, 2001), and were <6000 - 380,000 ng/L for D. Duplex 

(Goktepe and Plhak, 2002,2003). 

Glyphosate: Glyphosate was the lowest toxic (LC50 was 3042 ng/L) to M. micrura. Other 

values in reports were higher between 1150 - 107,000 (ig/L and 30000 Jig/L for C. Dubia 

and D. Magnaa, respectively. The LC50 value of pesticides showed that toxicity of 

chlorpyrifos > carbofuran > malathion > neem extract > glyphosate. M micrura were 

susceptible to pesticides from (ig/L to mg/L, with chlorpyrifos was the most toxic (LC50 = 

0.08 rig/L) and glyphosate was the lowest toxic (LC50 = 3042 Hg/L) to M. micrura. 

Pesticides 48 h-L50 ( u^L) 

Malathion 10.44(9.10-11.85) 

Chlorpyrifos 0.08 ( 0.03 - 0.20 ) 

Carbofuran 6.96 ( 5.97 - 7.63) 

Neem extract 196.3 ( 161.5 -263.9) 

Glyphosate 3043 ( 1974 - 1778) 

Table 2. Acute toxicity (Medium lethal concentration [LC50]) of pesticides on M. micrura at 
48 h. 



272 Pesticides in the Modern World - Risks and Benefits 

In this studied were founded that toxicity of the insecticide group (chlorpyrifos, carbofuran, 
malathion and neem extract) were more toxic to M. micrura than the herbicide group 
(glyphosate), because insecticide had mode of action that affect on organism directly but 
herbicide acted in indirect way. US. EPA (1998) reported chlorpyrifos had very high toxicity 
to freshwater fish and aquatic invertebrates, carbofuran and neem extract had higher 
toxicity but glyphosate had less toxicity on zooplankton (Henry et al.,1994; ENTOXNET, 
1996; PMRA, 2002; Dopsikova, 2003; Saglam and Saler, 2005). On the basis LCso value, M. 
micrura of this study were sensitive to pesticides nearly Ceriodapnia species but were more 
sensitive to pesticides than Dahpnia species indicated by other studies in Table 3. Due to 
Daphnia species were bigger than Moina species and Ceriodapnia species thus its tolerant 
than M. Micrura and Ceriodapnia species. The result were found similar to Scott and 
Kaushik (1998); Liane (2002) and Grant and Schmutter (1987) reported were size, age, 
species, life-cycle of zooplankton and environment such as temparature, pH and harness 
have influent to chemical toxicity on zooplankton. 

The observation of M micrura, after treated with pesticides especially in high concentration, 
the swimming activity of M. micrura was changed. They moved faster then normal 
conditions, after a time later, the movement on antenna and limbs become slowly and death 
after that. Concentration of pesticides had disrupt respiratory membrane of M. micrura, their 
swimming behavior changes in high concentration and M. micrura were loosed their original 
colored. The similar results were reported in Rassolzadegan (2000); Saglam and Saler 
(2005)John et al. (2007) 

Chronic toxicity 

Effect of sublethal pesticides concentration on the number of offspring per female of 
M. micrura is shown in (Table 4). Number of offspring per female of M. micrura was 
significant reduced (P<0.05) at malathion concentration 0.50 ng/L, chlorpyrifos concentation 
greater than 0.00025 ng/L, at carbofuran concentration at 2.50 Jig/L and at glyphasate 
concentration 325 ng/L. For neem extract concentration had no effect on the number of 
offspring per female significantly (P>0.05). Sublethal effects for each pesticide, were 
founded similar to other reports (Wong et al, 1995; Alberdi et al,1996; US EPA 2006). An 
estimate of no observed effect concentration (NOEC) and lowest observed concentration 
(LOEC) were 0.25 and 0.50 ng/L for malathion, 0.00005 and 0.00025 ng/L for chlorpyrifos, 
1.00 and 2.50 ng/L for carbofuran, 250 and 325 for glyphosate and LOEC 65 ng/L for neem 
extract. Cladocerans contribute an important component of aquatic ecosystem especially, for 
fish food source. If the number of clardocerans were down, it may affect fish and another 
organisms. 

The number of offspring per female is one endpoint used to determine the maximum 
acceptable - toxicant concentration (MATC). The 16 % reproduction impairment have been 
used as the endpoint for many aquatic ecotoxicology (Biesinger and Chistensen, 1972). 
Therefore, this studies used 16 % reproduction impairment estimate the chronic values 
MATCs for pesticides (Table 4). According to the obtained results the calculated values of 
MATCs and 48-h LC 50 were for estimate application factor (AF) of pesticides on M. micrura 
(Table 5). 

This value was used to predict the safe concentration (SC)applies for pollutant prevention in 
aquatic ecosystem. However, the application factor will vary with type of pesticide and 
organism (Mounth and Stephan, 1967). 



Using Zooplankton, Moina Micrura Kurz Evaluated 
Ecotoxicology of Pesticides Used in Paddy Field in Thailand 



273 





Pesticide 


Species 


48-h LC 50 


References 


Malathion 


M. micrura 


10.44 


This studies 




M. marcocapa 


5-10 


Wang et al. (1994) 




C. dubia 


3.18 


Hernadez et al. (2004) 






3.35 


Maul et al. (2006) 






1.14 


Nelson et al. (1997, 1998) 






2.12 


Ankley et al. (1991) 




D. magna 


0.90 


Ren et al. (2007) 






8.0 


Khan et al. (1993) 






13 


Siefirt. 1987) 


Clorpyrifos 


M. micrura 


0.08 


This studies 




C. dubia 


0.117 


Bailey. (1997) 






0.056 


Harmon et al. (2003) 




D. ambigue 


0.050 


El- Merhibi et al. (2004) 






0.035 


Harmon et al. (2003) 




D. magna 


0.30 - 0.80 


Caceres et al. (2007) 






1.28 


Van Wijngaarden et al. (1993) 




D. duplex 


1.0-3.7 


Barata et al. (2004) 






0.13 


Kersting et al. (1997) 






>1.6 


Van der Hoeven and Gerrisen. (1997) 






0.17-0.49 


Hooftmant et al. (1993) 


Carbofuran 


M. micrura 


6.96 


This studies 




C. dubia 


2.69 


Nerberg et al. (1997) 






>20 


Poirer (1990) 






>162 


DBR (2000) 




D. magna 


86.1 
38.6 
18.7 


Dopsikova (2003) 


Neem extract 


M. micrura 


196.3 


This studies 




D. magna 


1250 


John. (2001) 




D. duplex 


570 - 680 


Stark. (2001) 






570 


Scott and Kaushik. (2001) 






<6000 - 243000 


Goktepe and Plhak. (2002) 






30000 - 380000 


Goktepe and Plhak. (2003) 


Glyphosate 


M. micrura 


3043 


This studies 




C. dubia 


1150 


Hensen et al. (1994) 






5890 - 107000 


Tsui et al. (2004) 




D. spinulata 


30000 


Lutufu et al. (2001) 




D. magna 


20000 - 21880 


Al -Omar et al. (2000) 




D. duplex 


218000 


Henry et al. (1994) 






7900 


Office of pesticide program. (2000) 



Table 3. Comparison of 48-h LC50 ( Hg/L) Value of Moina micrura and another clardocerans 
species. 

Aquatic ecosystems in tropical regions differ from those in temperate regions. The 
biodiversity in tropical zones is higher than that in temperate zones, which means that in 
tropic regions there are potentially more species that can be exposed to certain pollutants. 
However, many countries in the tropics are developing countries, in which pollution control 



274 Pesticides in the Modern World - Risks and Benefits 

is not carried out due to a lack of funds and other resources. Furthermore environmental 
quality criteria for some pollutants are often obtained by extrapolating toxicity data derived 
for a reduced number of species mainly distributed in temperate regions (e.g. Europe or the 
US) (Kim et al, 2001 in Kwok et al, 2007). Kwok et al. (2007) investigated to which extent the 
sensitivity distributions of temperate species to toxic substances were similar to those of 
tropical species. They found that the temperate species seemed to be more sensitive to 
metals than the tropical species (Kwok et al, 2007). However, it should be noted that these 
differences might be due to the different species composition included in the species 
sensitivity distributions (SSD). Kwok et al (2007) used mainly fish species, which could be 
less sensitive to pollutants than the invertebrate species that are predominantly used in the 
temperate species sensitivity distributions. A better comparison can be made when using 
similar taxonomic groups for the distribution. 

In Thailand ecotoxicological research is quite new and has many limitations. Although 
ecotoxicological issues arise in this country and there is a need for water quality 
management and ecological risk assessment tools, there is a lack of ecotoxicological data on 
aquatic organisms from Thailand. Until now, like other developing countries, they have 
relied on over sea data to develop ecotoxicological test guidelines. However, these 
guidelines may be unsuitable for Thailand. The Thai indigenous aquatic organisms might be 
more or less sensitive to contaminants than their temperate surrogate species (Iwai, 2004; 
Iwai and Noller, 2010; Somparn et al., 2010). Moreover, there are differences in 
physicochemical and biological characteristics of aquatic habitats between tropical and 
temperate regions (Kwok et al., 2007). The characteristic of the sediment and water in Thai 
rivers may differ from those in other countries (Iwai and Noller, 2010; Somparn et al., 2010), 
influencing the concentration, availability and accumulation of pollutants and therefore 
their toxicity. An example of this is given by Jeon et al. (2010). They found that clay and food 
content in the water influence the toxicity of pollutants on aquatic biota. 
Tirado et al. (2008) report that the main rivers in Thailand were monitored from 1993 to 1999 
for the presence of pesticide residues; most water samples contained insecticide and 
herbicide residues in levels above advisable limits, whereas less contamination was 
observed in sediment samples. In river water, organochlorine pesticides were detected in 
40.62% of the samples (in concentration ranging from 0.01 to 1.21 ug/L), organophosphate 
pesticides were detected in 20.62% of samples (in concentration ranging from 0.01 to 5.74 
ug/L). The safety limit established by the European Union is 0.1 ug/L for any single 
pesticide and 0.5 ug/1 for the sum of all pesticides detected. Both organochlorine and 
organophosphate pesticide residues were found above those safety limits. Additional 
compounds, like carbamate pesticides were detected in 12.39% of samples (in concentration 
ranging from 0.01 to 13.67 ug/1), triazines were detected in 20.0% of samples (in 
concentration ranging from 0.01 to 6.63 ug/L), and paraquat was detected in 21.36% of 
samples (in concentration ranging from 0.14 to 87.0 ug/L) (Chulintorn et al., 2002). An 
earlier study has also found residues of the pesticides DDT and dieldrin in five Thai rivers 
(Upper Ping, Lower Ping, Wang, Yom, Nan, Chee), in concentrations above acceptable 
standard levels (Sombatsiri, 1997). The Division of Agricultural Toxic Substances in the 
Department of Agriculture (Ministry of Agriculture and Cooperatives) has also monitored 
the presence of pesticide residues in rivers and canals around agricultural areas in the 
country. The contamination of pesticides in water and sediments was generally low in water 
resources used for domestic consumption like ponds and reservoirs that have no connection 
to agricultural plantations. However, the water resources in certain agricultural areas, like 
orchid and ornamental plantations, were contaminated with organophosphate and 



Using Zooplankton, Moina Micrura Kurz Evaluated 

Ecotoxicology of Pesticides Used in Paddy Field in Thailand 275 

carbamate insecticides. From 1999 to 2001, a survey of three major rivers along paddy field 
areas (Thachin river in Suphanburi and Nakornpathom, the Chao Phraya river in 
Pathumthani and Nonthaburi, and the Bangpakong river in Chachengsao), found the 
highest residues of the insecticide endosulfan in the Thachin River, followed by the Chao 
Phraya and Bangpakong Rivers. In all cases, the levels of pesticide residues were above the 
safety limit set by the European Union (0.1 ug/L) (Chatsantiprapha, et. al., 2002). 
In 2001, groundwater in the lower Central and the lower Northeastern region of Thailand 
was contaminated with pesticides residues, in many cases in concentration above the safety 
limit set by the EU (0.1 ug/1). In the lower Central region during the rainy season in 2001, 
68% of 15 GRL-TN-03-2008 the total groundwater samples were contaminated with 
endosulfan and other insecticides, in concentration ranging from 0.02 to 3.2 ug/1, and 
paraquat, 2,4-D, butachlor, atrazine and metribuzin herbicide residues ranging from 0.02 to 
18.9 ug/1. In lower Northeastern region during the dry season in 2001, 71.2% of the total 
groundwater samples were contaminated with endosulfan and other insecticides, in 
concentrations from 0.01 to 0.33 ug/1, and atrazine and paraquat herbicide residues at the 
level of 0.5-4.0 ug/1 (Sakultiangtrong, et.al., 2002). In 1993, the Department of Agriculture 
investigated shallow groundwater wells from Rayong Province. From 160 samples collected 
from wells, 67% were contaminated with organochlorine and organophosphate pesticides, 
but in concentration below the safety limits (Pollution Control Department, 2004). 

Pesticide 

Malathion 



Chlorpyrifos 



Carbofuran 



Neem extrat 



Glyphosate 



Concentration 
(Ug/L) 


Number of offspring per female 


% Reproductive 
impairment 


0.00 


55.13±0.45a 


0.00 


0.05 


53.13±0.50a 


3.68 


0.25 


49.03±0.70a 


11.06 


0.50 


36.50±0.46b 


33.79 


0.00 


53.37+0.35a 


0.00 


0.00005 


53.37+0.35a 


6.741 


0.00025 


49.77±0.41b 


9.43 


0.00045 


43.00+0.52c 


28.23 


0.00 


53.37±0.35a 


0.00 


0.00005 


53.37±0.35a 


6.74 


0.00025 


49.77±0.41b 


19.43 


0.00045 


43.00±0.52c 


28.23 


0.00 


56.33±3.15a 


0.00 


15.00 


55.10+2.12a 


2.18 


40.00 


53.76+1.72a 


4.56 


65.00 


52.33+1 .99a 


7.10 


0.00 


56.06±1.62a 


0.00 


50.00 


55.17+0.95a 


1.59 


250.00 


47.66+2.12a 


14.19 


325.00 


42.43+3.74b 


24.31 



*Note:Value are maen + standard deviation. Mean with the same letter in the column are not 
significantly different (P>0.05). 

Table 4. Chronic toxicity of malathion, chlorpyrifos, carbofuran, neem extract and gyphosate 
on the number of offspring per female and % reproductive impairment of M. micrura. 



276 Pesticides in the Modern World - Risks and Benefits 





Pesticides 


48 h-LCso (Mg/L) 


MATC 


AF 


Malathion 


10.44 


0.36 


0.03 


Chlorpyrifos 


0.08 


0.0001 


0.001 


Carbofuran 


6.96 


2.41 


0.35 


Neem extract 


196.3 


281.89 


0.09 


Glyphosate 


3043 


172.04 


0.88 



Table 5. The maximum acceptable - toxicant concentration (MATC) and Application factor 
(AF) for each pesticide. 

4. Conclusion 

The aim of this study was using zooplankton, Moina micrura Kurz. which is an important 
species in aquatic ecosystem of Thailand to evaluated ecotoxicity of main pesticide used 
in paddy field (malathion, chlorpyrifos, carbofuran, neem extract (azadirachtin) and 
glyphosate). The acute toxicity (48-h LC50 ) of malathion, chlorpyrifos, carbofuran, neem 
extract and glyphosate on M. micrura were 10.44, 0.08, 6.96, 196.3 and 3043 ng/L, 
respectively. Chlorpyrifos had highest toxicity followed by carbofuran, malathion, neem 
extract and glyphosate, respectively. Chronic toxicity test, the effect of pesticides to M. 
micrura on reproduction was studies by observing the number of offspring per female. 
Reproduction have significant reduced (P<0.05), with concentration of malathion at 0.50 
Hg/L, chlorpyrifos greater than 0.0025 (ig/L, carbofuran at 2.50 (ig/L and the 
concentration of glyphosate at 325 ng/L affected on reducing the number of offspring per 
female significantly (P<0.05). The neem extract had no significantly (P>0.05) effect on the 
number of offspring per female. The maximum acceptable - toxicant concentration 
(MATCs) of malathion, chlorpyrifos, carbofuran, neem extract and glyphosate were 0.36, 
0.0001, 2.41, 172 and 281.9 jxg/L, respectively. The result would be useful as an input to 
developing a biomonitoring tool for evaluation pesticide contamination in Thailand 
aquatic ecosystem. 

Effect of experimental condition including duration test organism and end point on 
observed toxicity of pesticide to M. micrura were evaluated. Relative sensitivities of test 
varies with pesticide type. Among five pesticides toxicity test, chlorpyrifos had highest 
acute toxicity on M. micrura followed by carbofuran, malathion, neem extract and 
gyphosate, respectively. The significant reducing effect on number of offspring per female of 
M. micrura were observed in the present of malathion, chlorpyrifos carbofuran and 
glyphosate. For neem extract had no effect on the number of offspring per female. The 
results indicate that reproductivity parameters are very important interm of pesticide 
impact on aqutic population such as M. micrura. However, in the natural environment 
aquatic organism are often exposure to multiple pesticides simultaneously. Therefore under 
natural condition, there is the potential of pesticides may act in additive or synergistic 
manner, although the sensitivity of aquatic biota to multiple pesticides cannot be predicted 
by the individual pesticide sensitivities generate in this study. 

The results showed M. micrura to be sensitive test organism, Thus its a good bioindicator 
and useful to developing a biomonitoring tool for evaluation pesticide contamination in 
Thailand aquatic ecosystem. However, in order to obtain more precise and conclusive 
toxicology data on application of these pesticide in paddy field and evaluation toxicity of 
pesticides on organism, similar study using another local freshwater in Thailand. 



Using Zooplankton, Moina Micrura Kurz Evaluated 

Ecotoxicology of Pesticides Used in Paddy Field in Thailand 277 

5. Acknowledgment 

This work was supported by the Higher Education Research Promotion and National 
Research University Project of Thailand, Office of the Higher Education Commission. The 
authors wish to express their sincere thanks to Khon Kaen University for the research 
funding, Groundwater research centre (GWRC) and Graduate School Khon Kaen 
University. 

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03/2008.Greenpeace Research Laboratories. 
Tejada, A.W. (1995) Pesticides residues in foods and the environment as consequence of 

crop production. Philippine Agriculture, Vol.78,pp. 63-79 
Wong, C. K., Chu K. H. & Shum F. F. (1995). Acute and Chronic Toxicity of Malathion to 

the Freshwater Cladoceran Moina macrocopa. Water Air Soil Pollution, (November 

1994), Vol.84, No.3/4, pp. 399-405, ISSN 0049-6979 
Wongrat, L. (2001). Zooplankton. In: Faculty of Fisheries of Kasetsart University, (Ed.),pp. 787, 

Thailand, ISBN 9-745538412-4 
US. Environmental Protection Agency. (1993). Environmental fate and effects division. 

Pesticide environmental fate one line summary: Carbofuran. Washington, DC. 
US EPA. (1996). Ecological Effects Test Guidelines. In: OPPTS 850.1010 Aquatic Invertebrate 

Acute Toxicology Test Freshwater Daphnia. Department of Pesticide Regulation US 

Environmental 
US EPA. (1998). Registration Standard (Second Round Review) for the Reregistration of Pesticide 

Products Containing Chlorpyrifos. Office of Pesticide Programs, US EPA, Washington, 

DC 
US. Environmental Protection Agency. (2002). Short-Term Methods for Estimating the 

Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. In 

-.EPA-821-R-02-013, 4th edn, pp. 230,Cincinnati, OH 
US EPA. (2006). Interim Reregistration Eligibility Decision (IRED) Document for 

Carbofuran. In: EPA-738-R-06-031. Department of Pesticide Regulation US 

Environmental 
Van der Hoeven, N& Gerritsen, A. A.M. (1997). Effects of Chlorpyrifos on Individuals and 

Populations of Daphnia pulex in the Laboratory and Field. Environmental 

Contamination and Toxicology, (December 1997), Vol. 16, No.12, pp. 2438-2447, ISSN 

0730-7268 
Van Wijngaarden, R., P. Leeuwangh, W.G.H. Lucassen, K. Romijn, R. Ronday &. Van der 

Veld, R. (1993). Acute Toxicity of Chlorpyrifos to Fish, a Newt, and Aquatic 

Invertebrates. Bulletin of Environmental Contamination and Toxicology,Vol. 51, No.5, 

pp. 716-723, ISSN 0007-4861 



15 



Application of Some Herbal Extracts and 

Calcium as an Antidote to Counteract the Toxic 

Effects of Cypermethrin and Carbofuran in 

Indian Major Carp, Labeo Rohita 

Subhendu Adhikari*, Amita Chattopadhyay 1 and Biplab Sarkar 2 

1 Central Institute of Freshwater Aquaculture, 

2 KIIT University, 
India 



1. Introduction 

Pesticides that are transported to the aquatic environment are primarily of agricultural 
origin. Sometimes, pesticides are applied to the fish ponds to control fish diseases. In the 
process, the residues that reach the hydrosphere are concentrated in certain parts of the 
aquatic ecosystem or remain in solution for extended periods or adsorbed to the particulate 
matter and thereby deposited in the sediments. Thus, pesticides could be accumulated in the 
body of the aquatic animals. Most of the pesticides act on the respiratory process and 
cholinergic nervous system and hamper the cell metabolism in addition to other 
disturbances. Thus, a formulation of antidotes to counteract pesticides is an important 
aspect of pollution research and work in this direction is in the initial stages. Zamfir (1979) 
worked on the possibilities of removal of pesticide polluted water in treatment stations and 
described some methodologies, i.e., flocculation and filtration that can partially removed 
DOT, 2,4,5-T, Endrin, Parathion and Lindane. Chlorine oxidation can remove parathion; 
diuron etc., ozone and potassium permanganate appear to extract effects similar to those of 
chlorination. Activated charcoal has positive effects in the removal of absorption of most 
pesticides and U-V rays also can remove a certain amount of pesticides. 
Some indirect approaches have also been employed by some scientists and their methods 
were environmental or nutritional manipulation. Sado et. al. (1992) reported that increased 
temperature and optimum levels of dissolved oxygen (by aerator) can decrease the 
pesticidal action. The application of lime to increase the pH for counteraction of the toxic 
effects of pesticides is also documented. Ghazaly (1994) and Mukherjee (1996) evaluated 
efficacy of ascorbic acid (vitamin C) for the intoxication of different pollutants including 
pesticides. Application of different herbal extracts for this purpose could play a very 
important role to mitigate the toxic effect of pesticides. 



* Soil and Water Chemistry Section, Aquaculture Production and Environment Division, Central 
Institute of Freshwater Aquaculture, Kausalyaganga, Orissa, India 



282 Pesticides in the Modern World - Risks and Benefits 

2. Aim of the study 

To investigate some herbal extracts and water calcium as antidotes to counteract the 
pesticidal effects on Indian Major Carp, Labeo rohita (rohu). 

3. Materials and methods 

Three herbal extracts and calcium as possible antidotes were tried for their efficacy to 
counteract Cypermethrin,a synthetic pyrethroid (trade name-Cypermethrin, chemical name- 
Cyano methyl,2,2-dimethyl,cyclopropane-carboxylate) and Carbofuran, a carbamate 
pesticide (trade name-Furadan 3G, chemical name- 2,3 dihydro,2-2 dimethyl,7 benzo 
furymethyl carbamate) toxicosis in fish. Antidotes study was conducted in 2 steps (1) 
Preparation of Crude extracts and (2) Experimental trials. 

Datura (Datura ripens), Kalka (Nerium indicum) and Neem (Azadiricta indica) were the plants 
selected for antidote study and fruits of datura and kalka, and leaves of neem were the 
specific materials. These plant materials were minced, then grounded and finally extracted 
using acetone (50%) solution. The ratio of plant material and acetone solution was 
maintained at 1:10. The paste was collected in a fine meshed cloth and the filtrate was 
collected for the study. Everytime fresh extracts were prepared before trials. The doses of 
datura extract were 2.5 and 5.0 ml/1, for both the pesticides. The doses of kalka extract were 
2.5 and 5.0 ml/1, for the Cypermethrin experiment while the same were 0.75 and 1.5 ml/1, 
for the Carbofuran experiment. The doses of neem extract were 10 and 25 ml/1 for the 
Cypermethrin experiment while the same were 5.0 and 10 ml/1 for the Carbofuran study. 
For the application of calcium solution in water, calcium chloride was chosen as reagent. 
Fresh solutions were prepared before experiment and applied with different doses (50, 100, 
200, 300 and 400 mg/1 for both the pesticides under study) to the experiments. 
Rohu fingerlings (2.25±0.16g) were used for the antidote study. The fishes were collected 
from the institute's pond and acclimatized at laboratory condition for 15 days after treating 
the fish with 0.1% potassium permanganate solution. Probable lethal doses for both 
cypermethrin and carbofuran were detected by error and trial method in 40 litre plastic tubs 
containing 20 litres of water. The antidote study was also performed for both the pesticides 
in the same type of plastic tubs with 20 L of freshwater. The water used for this study was 
7.5 pH and 80 mg/1 total alkalinity as CaCC>3. Four treatments, viz., control (only fish, no 
pesticides, no antidote), fishes treated with only pesticide (lethal dose), fishes treated with 
only antidote and fishes treated with pesticide (lethal dose) and antidote solution, were 
maintained. In every case, antidotes were applied half an hour after the pesticide treatment. 
Two different doses of each antidote were tried for each of the pesticides. Three replications 
were maintained for all the treatments. For each case, sampling was done after 1, 2, 4, 6, 12, 
24, 48, 72 and 96 hours, respectively(if any mortality occurs), and observations were made 
and final data were calculated on the basis of comparison between the different 
experimental observations. The results were expressed as percentage of fish survivability. 
For significance of antidote study, one way ANOVA (Duncan Multiple Range Test) were 
done (Zar, 1974). Test of significance was examined at 5% level. 

4. Results and discussion 

Among the four doses of calcium, both at 100 and 200 mg/1 of water calcium levels, 67 
percent of fish survived up to 96 hours compared to 100 percent fish mortality at 50,300 and 
400 mg/1 concentration of water calcium against the lethal concentration of cypermethrin. 



Application of Some Herbal Extracts and Calcium as an Antidote to Counteract 

the Toxic Effects of Cypermethrin and Carbofuran in Indian Major Carp, Labeo Rohita 



283 



There was no significant difference in fish survivability between 100 and 200 mg/1 levels of 
water calcium (Table 1). Fifty percent of fish survivability after 96 hours of exposure with 
lethal concentration of carbofuran was recorded both at 100 and 200 mg/1 levels of water 
calcium while 100 percent of fish mortality was obtained with 50,300 and 400 mg/1 levels of 
calcium against lethal concentration of carbofuran up to 96 hours (Table 3). 









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Table 1. Effect of water calcium on the toxicity of cypermethrin to Labeo rohita under 
laboratory conditions 

The effects of neem, datura and kalka extracts as probable antidotes against the 
cypermethrin toxicity using fish survivability as indicator have shown in Table 2. Datura 
extract (10%w/v) at the levels of 2.5 and 5.0 ml/1 exhibited 37 and 50% fish survivability 
after 96 hours of exposure with lethal concentration of cypermethrin. The fish survivability 
between these two levels of datura extract showed a significant (P<0.05) difference. 
Hundred percent of fish mortality recorded both at neem and kalka extract after 96 hours of 
exposure with lethal level of cypermethrin. It may be mentioned here that at neem and 
kalka extracts, the fish died within 12 hours. 



284 



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under laboratory conditions. 



Application of Some Herbal Extracts and Calcium as an Antidote to Counteract 

the Toxic Effects of Cypermethrin and Carbofuran in Indian Major Carp, Labeo Rohita 



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conditions 

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toxicity using fish survivability as indicator are presented in Table 4. Datura extract (10% 
w/v) at the concentrations of 2.5 and 5.0 ml/1 showed 50 and 37 percent fish survivability 
after 96 hours of exposure with lethal concentration of carbofuran. The fish survivability at 
these two levels of datura extract exhibited a significant (P<0.05) difference. Hundred per 
cent of fish mortality was observed at all the concentrations of neem and kalka extracts with 
the exposure of lethal level of carbofuran. It is important to note that the fish died within an 
hour at neem extracts while at kalka extracts, the fish died within 4 hours against the lethal 
level of carbofuran. 

The toxic effects of both cypermethrin and carbofuran decreased at medium level (100-200 
mg/1) of calcium of water. The results indicate that the degradation of these insecticides take 
place at medium calcium level of water. The decreased toxicity at this level of calcium could 
be associated with accumulation of insecticides in excess amount which may be metabolized 
and stored in different tissues. The accumulated insecticides are eliminated through urine of 
faeces or both with the help of liver, intestine and kidney (Subbiah et al., 1985). Metz and 



286 



Pesticides in the Modern World - Risks and Benefits 





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Table 4. Efficacy of some antidotes to carbofuran on survivability (%) of Labeo rohita under 
laboratory conditions. 

Branacin (1975) reported that the degradation of endosulfan took place at high pH and 
hardness of water but in case of triazophos, its toxicity was increased in similar conditions 



Application of Some Herbal Extracts and Calcium as an Antidote to Counteract 

the Toxic Effects of Cypermethrin and Carbofuran in Indian Major Carp, Labeo Rohita 287 

while Khalid (1985) reported that water hardness had no significant affect on endosulfan 
toxicity to four species of fish. Synthetic pyrethroids and natural pyrethrums were more 
toxic in hard water (Mauck et al., 1976). 

From the present study, it is clear that both the neem and kalka extracts had no antitoxic 
effects against cypermethrin and carbofuran poisoning, while acetone extracts of datura 
served as anti-toxicant against cypermethrin and carbofuran poisoning. The medicinal and 
therapeutic roles of datura fruit and its seeds have already been established. The young 
fruits are sedative and slightly intoxicating. The seeds are antispasmodic, narcotic, febrifuge, 
anthelmintic, works well in inflammation, alexiteric, emetic and useful in leucoderma, 
ulcers, itching, etc. The seeds contain both hysocyamine and scopolamine (Kirtikar and 
Basu, 1935). These medicinal qualities have a positive role for the survivability of pesticide 
intoxicated fish but the specific causes behind it is unknown and its need further 
investigation. However, it may be due to narcotic and intoxicated properties of datura 
which may have a neutralizing action against pesticide contamination. 

It can be mentioned here that vitamins are also having antitoxic effects against insecticide 
poisoning. For example, vitamins like Macraberin forte (a mixture of vitamins) served as 
antitoxicant against malathion poisoning to C. punctatus (Vaid and Mishra, 1977). When 
Channel catfish were exposed to various amounts of toxaphene (an organochlorine 
insecticide) in the diet for 150 days, a dose dependent depression of backbone collagen and 
spiral deformities were occurred but it recovered effectively when ascorbate (vitamin C) was 
added to the diet. Vitamin C supplements also reduced the whole body residues of 
toxaphene (Mayer et al., 1978). 

5. Conclusion 

From the present investigation, it is evident that though, pesticides induced changes of the 
fish could be improved/protected to a great extent by the application of acetone extract of 
datura and liming (calcium) up to a certain extent, further investigations are necessary in 
this regard. Therefore, judicious application of carbofuran in the paddy field to control rice 
pest and cypermethrin in the pond to control mosquito eggs and Argulus of fish are highly 
essential for sustainable growth of aquaculture. 

6. References 

Ghazaly, K.S. (1994). Efficacy of ascorbic acid (vitamin C) on experimental copper 

intoxication in Tilapia Zilli. Bull. Natl. Inst. Oceanogr. Fish., Egypt. 20(2):249-257. 
Khalid, M.S. (1985). Effects of pesticides on freshwater fish, Rasbora daniconius. Ph. D. Thesis, 

Marathwada University , India. 
Kirtikar and Basu (1935). Indian Medicinal Plants. 
Mayer, F.L., Mehrle, P.M. and Cruteher, P.L. (1978). Interaction of toxaphone and vitamin C 

in Channel catfish. Trans. Am. Fish. Soc, 107:326. 
Metz, J. and Branacin, M. (1975). Biochemical and biophysical aspect of salt excretion by 

chloride cell in teleost. In: Excretion (A.Wessing, Ed.), pp. 127-135. 
Mauck, W.L., Olson, L.E., and Marking, L.L. (1976). Toxicity of natural pyrethrins and five 

pyrethroids to fish. Arch. Environ. Contam. Toxicol., 4: 18. 



288 Pesticides in the Modern World - Risks and Benefits 

Mukherjee, M. (1996). Antidote to combat white spot disease in culture ponds. Fish.Chimes, 

16(5):30-31. 
Sado, E.K. and Ita, E.O.(1992). The effects of capture methods, temperature and oxygen on 

the survival of live freshwater clupeids during acclimation and transportation. J. 

Aquacult. Trop., 7(2):165-176. 
Subbiah, G.N., Marimathn,K. and Kamla, S.M. (1985). A study on the biological 

magnification of insecticide endosulfan in tissues of a fresh water fish, Tilapia 

mossambica. Proc. Symp. Asserds. Environ. Pollut., p.199-204. 
Vaid, S. and Mishra, I.M. (1997). Impact of Macraberin Forte against malathion poisoning in 

fish brain. Poll. Res., 16(4): 275-276. 
Zamfir, Gh. (1979). Water pollution by pest controlling agents and the possibilities for their 

removal in surface water treatment stations. Igiena, 28(2):97-103. 
Zar, J.H. (1974). Bio statistical Analysis, Prentice Hall, New Jersey, pp.260. 



16 



Semi Aquatic Top-Predators as Sentinels of 

Diversity and Dynamics of Pesticides in Aquatic 

Food Webs: The Case of Eurasian Otter 

(Lutra lutra) and Osprey (Pandion haliaetus) 

in Loire River Catchment, France 

Charles Lemarchand 1 , Rene Rosoux 2 and Philippe Berny 1 

toxicology laboratory, VetAgro Sup, Marcy I'Etoile, 
2 Museum des Sciences Naturelles d 'Orleans, 

France 

1. Introduction 

The Eurasian otter (Lutra lutra, Lutrinae, figure 1) and the osprey (Pandion haliaetus, 
Pandionidae, figure 2), formerly widespread in Europe and in France, have strongly declined 
during the 20th Century, following direct persecutions, habitat alteration and pollution, and 
consecutively decline of main prey. Direct persecutions were perpetrated because both 
species were considered as active competitors for fishing activity: otters were massively 
trapped for fur, and osprey populations were dismantled by direct shot or egg destruction 
in nests. Otter and osprey are both semi-aquatic top-predators species: diet is highly 
dominated by fish, which constitute at least 80 % and almost 98-100 % of the averaged prey 
biomass consumed by otter and osprey, respectively. However, diet studies of otter and 
osprey never showed any strong predation impact on fish diversity or biomass in rivers 
(reviews in Poole, 1989; Thibault et al. 2001; Clavero et al. 2003; Britton et al. 2005; Kruuk, 
2006; Dennis, 2008). This diet specificity influence otter physiological characteristics: by 
comparison with other mammalian carnivores, a specific diversity and accumulation pattern 
of essential fatty acids of aquatic origin from food to otter tissues was recently shown 
(Koussoroplis et al. 2008). Another diet characteristic of both species is the diversity of prey 
and their opportunistic hunting behaviour: almost all fish species available in otter or 
osprey local habitat are able to be consumed, depending on hunting conditions, with 
important diet variations between seasons, during life cycle or between populations. This is 
particularly true concerning osprey, a migrating species present in northern and western 
Europe during reproductive season, and wintering from southwestern Europe to sub- 
Saharan Africa, but was also observed concerning sedentary otter. Because of their high 
trophic level, habitat requirements and main ecological characteristics, otter and osprey can 
be considered as good sentinels and indicator species of global contamination and 
biomagnification of toxic contaminants in aquatic food webs of large rivers, estuaries, 
reservoirs and lakes. 



290 



Pesticides in the Modern World - Risks and Benefits 




Fig. 1. Eurasian Otter (Lutra Intra, adult male, photo R. Rosoux). 

Indeed, after direct destructions, contamination by persistent pollutants (e.g. pesticides, 
polychlorinated biphenyls (PCBs) or heavy metals) is blamed to be the causative agent of the 
decline of otter and osprey populations, throughout Europe as elsewhere in the world. On 
seldom occasions, acute poisoning after direct exposure or secondary poisoning by ingestion 
of highly contaminated prey were observed on otters in France, especially after oil spills, 
other industrial accidents or following heavy treatments with insecticides, avicides or 
rodenticides (Fournier-Chambrillon et al. 2004; Lafontaine et al. 2005; Berny, 2007; 
Lemarchand et al. 2010). Elsewhere in the world, acute poisoning of many marine otters 
(Enhydra lutris) were reported after Exxon Valdez oil spill in 1989 in Alaska (Garshelis and 
Jonhson, 2001). On the other side cases of acute poisoning were very rarely observed on 
ospreys. Potential long-term effects of trophic originating toxic compounds (i.e. chronic 
poisoning) on otters and ospreys were often investigated through reports of contamination or 
intoxication cases on monitored populations. Pesticides, and particularly organochlorine 
(OC) pesticides were the most commonly analyzed elements (but also heavy metals and 
PCBs, these latter generally associated with OC pesticides to assess the total OC 
contamination). Pesticides uses in the European Union are clearly specified by Directive EC 
91/414, but illegal poisoning of wild, game and domestic animals still occurs, and often 
results from pesticides abuse or illegal use (Berny, 2007; Berny and Gaillet, 2008). Among 
OC pesticides, dichorodiphenyltrichloroethane (DDT) and its main metabolites, particularly 
dichlorodiphenyldichloroethylene (DDE), were shown to accumulate in otter and osprey, 
causing body condition alteration, direct reproductive failure (like eggshell thinning 
observed on osprey) and consecutively population decline (Spitzer et al. 1978; Wiemeyer et 
al. 1988; Mason and Macdonald 1993a, b; Ewins et al. 1999; Elliott et al. 2000; Ruiz-Olmo et 
al. 2000; Henny et al. 2008). As these deleterious effects were documented on a very large 
spectrum of wild and domestic species after insect pests control, DDT uses were severely 
controlled or banned from the 1970's in developed countries (ban from 1973 in France). 
Nevertheless, DDT is still used in developing countries, particularly in India or Africa by 
indoor spraying during anti-malaria campaigns. The risk of DDT flow into agricultural and 



Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic 

Food Webs: The Case of Eurasian Otter (Lutra lutra) and Osprey (Pandion haliaetus) in Loire ... 291 

aquatic systems or wild environment of these countries was recently underlined (UNEP, 
2008). Due to environmental stability and persistence of DDT and metabolites, and probably 
following post-ban use of old stocks, these compounds are still present in environment and 
were recently detected in otters or ospreys (Kannan et al. 2004; Lemarchand et al. 2007, 2010; 
Henny et al. 2008). For this latter species, continuing of DDT use in some developing 
countries is an additional threat during the wintering period. Lindane, and to a lesser extent, 
Aldrin, Endosulfan and Methoxychlor were the main other OC pesticides quantified in otter 
or osprey during previous studies. However, most of studies reported low concentrations of 
these compounds when compared to DDT residues, and therefore a limited contribution to 
total OC contamination (Wiemeyer et al. 1988; Mason and Macdonald, 1993a,b, 1994; Elliott 
et al. 2000; Rattner et al. 2004; Lemarchand et al. 2010). 

Cholinesterase inhibitors as organophosphate (OP) and carbamate (CA) pesticides were 
widely used worldwide as insecticides for the protection of cultivated plants or livestock. 
Direct and indirect toxicity of cholinesterase inhibitors (e.g. Carbofuran, Mevinphos) were 
underlined on insect-consumer birds, birds of prey and scavengers like white-tailed sea 
eagle (Haliaetus albicilla) or red kite (Milvus milvus) (Hart et al. 1993; Elliot et al. 1996; Berny 
and Gaillet, 2008). These insecticides are highly toxic to birds of prey, nevertheless 
intoxication cases following OP and CA pesticides contamination remained rare when 
compared to total reported deaths (Fleischli et al. 2004). Pyrethroids insecticides were 
recently preferred to OP and CA pesticides uses. Indeed, pyrethroids pesticides are 
considered as a safer method of pest control because of their lower direct toxicity on 
mammals and birds (Martin et al. 1998; Chu et al. 2005). Nevertheless, data on pyrethroids 
insecticides diversity, persistence or toxicity in wild fauna are very poor in literature 
concerning top predators species. 




Fig. 2. Osprey (Pandion haliaetus, adult female; photo C. Lemarchand) 

Related to their plant-specific metabolic action, water solubility and poorly lipophilic 
characters, herbicides are documented as less toxic pesticides to vertebrates than insecticides 



292 Pesticides in the Modern World - Risks and Benefits 

(Berny, 2007). Data on herbicides diversity and toxicity on vertebrates or predators, especially 
otters or ospreys, are particularly rare. Nevertheless, some studies demonstrated a direct effect 
of herbicides on herbivorous mammals or bird diversity or abundance during land use 
modifications (Santillo et al. 1989a,b). Bioaccumulation potential of herbicides to a top predator 
was recently confirmed by a study in Washington State (USA) on sediments, fish and ospreys 
(Chu et al. 2007). Furthermore, recent studies underlined a direct impact of herbicides, 
particularly triazines, on fish and amphibians' reproduction or survival (Langlois et al. 2009; 
Tillitt et al. 2010). Direct impact of herbicides on fish or amphibians' populations would 
indirectly affect otters and ospreys by a reduction in food resource. Therefore toxicity of some 
persistent herbicides on vertebrates could be underestimated by an insufficient risk evaluation. 
At the beginning of the 1980's in France, otters only survived in two distinct populations: in 
the Massif Central mountains (centre), and along Atlantic Ocean and western wetlands of the 
country (Bouchardy, 1986). At the same period, osprey had disappeared of continental France 
as a nesting species. Legal protection of the otter and the osprey was decided from 1976. First 
signs of species recovery or return were recorded soon after. From 1985 increase and 
expanding of otter populations were proved and monitored in the whole repartition area of 
the species in France (Bouchardy, 1986; Rosoux and Bouchardy, 2002). In spring 1984, one pair 
of osprey stopped its migration towards northern Europe and built a nest along Loire River. 
Species is nesting again in continental France since 1985 (Coll., 1996). As osprey is a semi- 
colonial and philopatric species, other pairs quickly mated close to the first one, starting a new 
expanding population. European directives and national action plans allowed the protection 
and / or the restoration of both species habitat (Rosoux et al, 1999; Nadal and Tariel, 2008; 
Kuhn, 2009). The main characteristic of these species recoveries is their entirely natural 
process. Indeed, otter and osprey were never been reintroduced or reinforced in France, in 
order to establish habitats requirements, main natural and anthropogenic limits to 
populations, to locate colonization corridors and major sites for reproduction and breeding. 
After about three decades of protection, otter population in France is still increasing, formerly 
isolated populations met from the beginning of the 2000's and the repartition area of the 
species covers the whole Massif Central related to the western third of the country (Bouchardy 
et al. 2001; Kuhn, 2009; Lemarchand and Bouchardy 2011). 37 reproductive pairs of ospreys 
wee noted in 2010 in continental France, mainly distributed along the medium part of the 
Loire River, but a geographical expansion of the species towards other river systems was 
recently noted (Nadal and Tariel, 2008). Increase of otter and osprey populations particularly 
concerns Loire River catchment, a major dispersal corridor that should be decisive for species 
conservation and dispersion in the whole country. As many predators, otter and osprey 
suffered from a bad reputation, but are now associated with preserved habitats and food 
resource (Chanin, 2003; Whitfield et al. 2003; Grove et al. 2009). However, otter and osprey 
remain sparse in France and are listed on UICN National Red Lists as "Minor preoccupation 
(LC)" and "Vulnerable (VU)", respectively (UICN France et al. 2008, 2009). 
Objectives of this study were to evaluate the contamination of two flagship species 
(European otter and osprey) by a wide spectrum of pesticides, using a standard protocol of 
pesticides analyses in wild or domestic fauna and a non-invasive animal approach during a 
natural recolonization process in Loire River catchment. Since 2004 for the otter and 2007 for 
the osprey, a large toxicological program was launched during the "Plan Loire Grandeur 
Nature" program in France. 45 pesticides, including herbicides, organochlorine, 
organophosphate, carbamate and pyrethroids pesticides and a few main metabolites were 
systematically analyzed in otters and ospreys (but also in great cormorants, freshwater fish 
and invertebrates) from Loire River catchment (Lemarchand et al. 2007, 2009, 2010). 



Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic 

Food Webs: The Case of Eurasian Otter (Lutra lutra) and Osprey (Pandion haliaetus) in Loire ... 293 

2. Materials and methods 

2.1 Study area 

The study area corresponded to the whole Loire River and main tributaries catchment in 
France (Fig. 3). Loire River catchment (117000 km 2 , total length of rivers and tributaries of 
about 40000 km) is characterized by an important diversity of habitats and species, and is 
considered as one of the most preserved large hydrosystems in Western Europe. A national 
and European action plan, "Plan Loire Grandeur Nature", is running since 1994 to study 
and conserve this diversity, but also to protect inhabitants from floods and to maintain 
economic activity. 




Fig. 3. Map of the Loire River (bold) catchment in mainland France 

2.2 Animals monitoring and sampling 

Concerning such rare species, it is particularly difficult to obtain sufficient sample material 
from enough individuals to support analysis and statistics. For ethical reasons it was not 
imaginable to trap or kill otters or ospreys for analyses. To avoid any vital risk related to 
handling, capture and bleed of animals were not considered. Furthermore, otter and osprey 
are fully protected by national and international laws, and listed as species of interest by the 
European Community (Habitats Directive 92/43/EC, Birds Directive 79/409/CEE). All 
operations were therefore entirely conducted under appropriate authorizations by a non- 
invasive approach. A large network, constituted by people in charge of otter and osprey 
studying and monitoring in mainland France was built to organize and enhance sampling 
under the coordination of the Museum d' Orleans. The national agency for game and wildlife 
(ONCFS), hunting federations (FDC), the national agency for water and aquatic environments 
(ONEMA), health centres of the national union (UFCS) and of the birds protection league 
(LPO - French representative of Bird Life International), the national research centre on birds 
population biology (CRBPO, associated with the French national museum of natural history 
MNHN and Mr Rolf Wahl, in charge of osprey ringing program in France), the French 
Ministry of Environment (MEEDDM and DREAL Centre), the national agency for forests 
(ONF), private land owners and companies, museums, associations ("Loiret Nature 
Environnement") and regional naturalists were contributors for this study. 
Concerning otters, only road-traffic killed individuals and those found dead in the wild in 
study area were collected. Based on visual observation, carcasses found more than 24h 
(during summer) or 48h (during winter) after road collision were considered too degraded 



294 Pesticides in the Modern World - Risks and Benefits 

and not taken into account for post-mortem examination and toxicological analyses. 
Concerning ospreys, non-hatched eggs and dead young in nests were collected during 
chicks ringing operations. As scientists and birdwatchers monitor a majority of osprey nests 
in continental France, non-hatched eggs and dead young in nests were reported and 
sampled as soon as possible. France is also a major crossing area for migrating osprey from 
different populations (Hake et al. 2001; Dennis, 2008; Strandberg et al. 2009). Due, in one 
way, to the extreme rarity of this species in continental France (less than one hundred 
reproductive individuals), and in an other way that "foreigners" individuals (i.e. born in 
neighbour countries, but potentially breeders in France) are able to be found dead within the 
national territory (naturally or after illegal shots, electrocution on power cables, or drown in 
fish farms), migrating individuals flying towards reproduction areas elsewhere in Europe 
(Germany, Great-Britain, Scandinavia) completed sampling. 

All samples were deep-frozen (-40 °C) prior to analyses. For each otter or osprey carcass, a 
necropsy was performed, and about 20 g of liver was sampled. This organ was preferred to 
fat because some otters and a lot of ospreys have very little fat, particularly at the end of 
spring migration concerning these latter. Otter sex and weight were determined; animals 
were measured (total and head, body, foot and tail lengths). Age was defined as "juvenile" 
(milk teeth, little size and weight), "subadult" (adult size and weight, teeth without wear 
and tartar) and "adult" or "old" (worn teeth with tartar). Body condition index K was 
determined according to Kruuk and Conroy (1991). Osprey sex and weight were 
determined; animals were measured (wing, body, foot and tail lengths). Non-hatched 
osprey eggs were drilled and emptied; eggshell was conserved for future studies on shell 
thickness. Age was defined as "egg" (non-hatched), "juvenile" (non-flying hatched 
individual), "subadult" (emancipated individual with the characteristic creamy fringe on 
feathers) and "adult" (adult size and plumage) (Dennis, 2008). Each animal (otter or osprey) 
is characterised by a specific case-record gathering discovery circumstances, clinical and 
biometrical data. After necropsies, carcasses were conserved for further showing or 
collection in museums or, if too degraded, systematically destroyed according to law. 

2.3 Choice of compounds 

Pesticides uses in France are one of the biggest in the world. Various compounds have been 
used for wood, vineyards, orchard, crops or ornamental plant protection, human or 
livestock health, and roads, railways or boat maintenance. Origins and flow of compounds 
are complex and aquatic habitats are exposed to both direct and indirect contamination. 
In such a generalist approach, the choice of analyzed compounds is crucial and has to be 
representative: 

Of the diversity of uses (agrochemicals, industrials or domestics) in study area, 

Of the available analytical techniques and limits, 

Of accumulation and transfer patterns from trophic webs components to studied 

species. 
A specific detection and quantification methodology was developed for pesticides in the 
toxicology laboratory of the college of veterinary medicine (VetAgro Sup, Lyon, France) 
during routine analyses on wild, game or domestic fauna. Compounds were chosen 
according to their toxicity on fauna, persistence in soil and water and accumulation in food 
webs. Regular complements and upgrades were added, as a function of new compounds or 
new detection techniques. Detected compounds are listed in Table 1 below. 



Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic 
Food Webs: The Case of Eurasian Otter (Lutra lutra) and Osprey (Pandion haliaetus) in Loire . 



295 





Pesticides 


Pesticides family and main use 


Molecular formula 


Date of ban in France (or 
current status) 


Lindane (gamma-HCH) 


Organochlorine insecticide 


C 6 H h Cl6 


1998 


Endosulfan 


Organochlorine insecticide 


OHtCUOsS 


2007 


DDT 


Organochlorine insecticide 


C14H9CI5 


1972 


Heptachlor 


Organochlorine insecticide 


C 10 H 5 Cl7 


1973 


Aldrin 


Organochlorine insecticide 


Ci 2 H 8 CI 6 


1992 


Methoxychlor 


Organochlorine insecticide 


C 16 H 13 C1 3 2 


2002 


Methiocarb 


Carbamate insecticide, 
molluscicide 


C11H15NO2S 


Still in use 


Carbofuran 


Carbamate insecticide 


C12H15NO3 


2008 


Mevinphos 


Organophosphate insecticide 


C7H13O6P 


2004 


Phorate 


Organophosphate insecticide 


C7H17O2PS3 


2004 


Dichlorvos 


Organophosphate insecticide 


C4H7C12O4P 


2007 


Terbufos 


Organophosphate insecticide 


C9H21O2PS3 


2004 


Diazinon 


Organophosphate insecticide 


C12H21N2O3PS 


Still in use 


Disulfoton sulfone 


Organophosphate insecticide 


C8H19O2PS3 


2004 


Chlorpyriphos ethyl 


Organophosphate insecticide 


C9H11C13NO3PS 


Still in use 


Fenitrothion 


Organophosphate insecticide 


C9H12NO5PS 


Still in use 


Pyrimiphos methyl 


Organophosphate insecticide 


C11H20N3O3PS 


Still in use 


Malathion 


Organophosphate insecticide 


C10H19O6PS2 


2008 


Fenthion 


Organophosphate insecticide 


C10H15O3PS2 


2005 


Parathion 


Organophosphate insecticide 


C10H14NO5PS 


2002 


Methidathion 


Organophosphate insecticide 


C6H11N2O4PS3 


2004 


Triazophos 


Organophosphate insecticide 


C12H16N3O3PS 


1992 


Trifluraline 


Anilide herbicide 


C13H16F3N3O4 


2008 


Atrazine 


Triazine herbicide 


CsHuCINb 


2003 


Simazine 


Triazine herbicide 


C7H12CLN5 


2003 


Terbuthylazine 


Triazine herbicide 


C9Hi 6 aN 5 


2003 


Cyanazine 


Triazine herbicide 


C9H13C1N6 


2004 


Alachlor 


Chloroacetanilide herbicide 


C14H20C1NO2 


2008 


Metolachlor 


Organochlorine herbicide 


C15H33C1N02 


2003 


Diuron 


Substituted phenylurea 
herbicide 


C9H10C12N2O 


2008 


Epoxyconazole 


Fongicide 


C17H13C1FN30 


Still in use 


Tefluthrine 


Pyrethroid insecticide 


C17H14C1F702 


Still in use 


Cyhalothrine Lambda 


Pyrethroid insecticide 


C23H19C1F3N03 


Still in use 


Permethrine Cis 


Pyrethroid insecticide 


C21H20CI2O3 


Still in use 


Cyfluthrine 2 


Pyrethroid insecticide 


C22H18C12FN03 


Still in use 


Cypermethrine 2 


Pyrethroid insecticide 


C22H19CL2N03 


Still in use 


Fenvalerate Cis 


Pyrethroid insecticide 


C25H22C1N03 


Still in use 


Deltamethrine 


Pyrethroid insecticide 


C22Hi9Br 2 N0 3 


Still in use 



Table 1. List of families and uses, molecular formulae, current status of the compounds 
analyzed in this study. 



296 Pesticides in the Modern World - Risks and Benefits 

2.4 Pesticides quantification methods 

2.4.1 Organochlorine pesticides 

2.0-8.0 g of tissue were sampled and 30 ml of hexane/acetone 75/25 mix was added. Each 
sample was blended twice with an Ultraturrax® (Ika, Werke, Germany) and filtered trough a 
phase separator membrane. The extract was evaporated at 60 °C in a rotary evaporator. The 
dry extract was dissolved in 10 ml hexane. 

Two ml of fuming sulphuric acid (S0 3 7%) were added, and after centrifugation at 4x g, 1 ml 
of the supernatant was used for OC pesticides quantification by gas chromatography with 
electron capture detection material. Temperature program and injection conditions are 
described in Lemarchand et al. (2007; 2010). Each sample was run in duplicate. 
Organochlorine pesticides concentrations were calculated by using different mix standards. 
Recovery level on standard mixtures was always greater than 92%. All standards were 
purchased from CIL (St Foy la Grande, France), and purity was > 99%. Linearity was 
determined between 5 and 100 ng.g- 1 (r 2 > 0.99 on standards and spiked samples, 5-point 
calibration curves). Limits of detection were between 0.5 and 1.0 ng.g- 1 lipids for individual 
PCB congeners. Cod liver oil (BCR349) certified material was used as a regular quality 
control. 

2.4.2 Organophosphate pesticides analyses 

5 g of muscle sample was shaken with 60 ml dichloromethane and 10 g anhydrous sulfate. Mix 
was then filtered trough a Whatman 1 PS membrane, and evaporated under vacuum at 40°C. 
Dry samples were diluted in 3 ml ethanol, and underwent an ultrasonic step. Extract was then 
purified with a Sep pack R 300 (Silica Waters, 020810; 500 mg) column conditioned with 2 ml 
methanol and 2 ml ethanol. 2 ml dichloromethane were used for column elution. Purified 
samples were dried and diluted with 3 ml dichloromethane. Organophosphate (OP) and 2 
carbamates (CA) pesticides (Dichlorvos, Carbofuran, Mevinphos, Phorate, Phorate oxon, 
Phorate sulfone, Methiocarbe, Terbufos, Diazinon, Disulfoton, Chlorpyriphos methyl, 
Chlorpyriphos ethyl, Fenitrothion, Pyrimiphos methyl, Malathion, Fenthion, Parathion, 
Methidathion, Disulfoton sulfone, Triazophos) concentrations were determined by GC/MS in 
SIM mode (OP + carbofuran and methiocarbe). A 5973N MS coupled with a 6890 GC 
(Agilent®) was used, with a 30m HP5-MS column (0.25 mm ID, 0.25jim thickness). For each 
samples standard and spiked sample, 2 jiL were injected. The temperature program was 100°C 
(2 min), 55°C/min up to 200 °C (held for 5 min), 50 °C up to 220 °C (held for 3 minutes), 
followed by 60 °C/min up to 300°C. A final, post-run time of 2 min at 300°C was maintained. 
Total run time was 13.55 min. Injector was set at 250°C and the He flow was set at 2.5 ml/ min. 
Each OP or CA was identified based on the following criteria: retention time and 3-4 
fragmentation ions with pre-defined relative amounts and 20% variability acceptance for each 
ion. Linearity was confirmed between 25 and 500 ng.g- 1 with 5 point calibration curves and r 2 
>0.99. Recovery was determined between 76% and 104% for all spiked samples and 
repeatability was considered acceptable with coefficients of variation <15% . 

2.4.3 Pyrethroids pesticides analyses 

5 g of tissue (liver or muscle) sample was shaken in 60 ml ethanol and 10 g anhydrous 
sulfate, and then filtered trough a Whatman 1 PS membrane. Extract was dissolved in 5 ml 
methanol and underwent a second filtration procedure. Concentrations were determined by 
GC / ECD and confirmed by GC/MS according to a modified method of the French Food 
Safety Authority (Anses Met AFSSA). An Agilent GC-ECD 6850 with a 30m HP1 column 



Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic 

Food Webs: The Case of Eurasian Otter (Lutra lutra) and Osprey (Pandion haliaetus) in Loire ... 297 

(0.32 mm ID, 0.25jim film) was used. For each samples standard and spiked sample, 2 uL 
were injected. The temperature program was common to OCs', PCBs and pyrethroids 
(initial temp: 100°C, first ramp 6°C/min up to 220 °C held for 10 min, 2 nd ramp 7 °C/min up 
to 285°C, held for 1 min, total run time 42.29 min) Injector was at 230°C, detector at 300°C. 
Total He flow was 9 ml/ min. Pyrethroids were identified according to their retention times. 
Linearity was confirmed between 10 and 100 ng.g- 1 with 5 point calibration curves and r 2 
>0.99. Recovery was determined between 82% and 94% for all spiked samples and 
repeatability was considered acceptable with coefficients of variation <15%. For all positive 
samples, a confirmatory analysis was performed with GC/MS in SIM mode. Identification 
was based on retention times and 3 or 4 ions. 

2.4.4 Herbicides analyses 

2 g of muscle sample was shaken during 5 minutes in 8 ml acetone, and then centrifuged at 
4x g; supernatant was placed in separate tubes, and this extraction was performed twice. 
Samples were evaporated under nitrogen, and dry extract was dissolved in 1 ml 
aceton/ methanol (50:50) solution. Extract was then purified with a SPE C18 500 mg column 
conditioned with 2 ml acetone and 2 ml methanol. Column was vacuum dried and purified 
samples were diluted in 3 ml acetone. After drying under nitrogen, samples were diluted in 
1 ml methanol. Herbicides (Trifluraline, Atrazine, Simazine, Terbuthylazine, Diuron, 
Alachlor, Metolachlor, Cyanazine, Epoxyconazloe) concentrations were determined by 
GC/MS spectrometry. A 5973N MS coupled with a 6890 GC (Agilent®) was used, with a 
30m HP5-MS column (0.25 mm ID, 0.25|im thickness). For each samples standard and 
spiked sample, 2 j^L were injected. The temperature program was 85°C held 1 min, followed 
by 6°C/min up to 170°C (held for 12 min), then followed by 20°C/min up to 280°C, held for 
4.33 min (total run time 37 min). Injector was at 250 C C and in the splitless mode. Each 
herbicide was identified based on the following criteria: retention time and 3-4 
fragmentation ions with pre-defined relative amounts and 20% variability acceptance for 
each ion. Linearity was confirmed between 100 and 500 ng.g- 1 with 5 point calibration 
curves and r 2 >0.99. Recovery was determined between 67% and 98% for all spiked samples 
and repeatability was considered acceptable with coefficients of variation <15% . 

2.5 Calculation methods and statistical analysis 

Geometric means of p,p'-DDE, p,p'-DDD and p,p'-DDT were added to calculate the sum of 
DDTs (Z DDTs). Geometric means of lindane, endosulfan, DDE, DDD, DDT, heptachlor, 
heptachlor epoxyde, aldrin and metoxychlor were summed to provide the sum of pesticide 
concentrations (Z Pesticides). All these were chosen by the National Veterinary School of 
Lyon (VetAgro Sup, France) standard protocol (Mazet et al. 2005; Lemarchand et al. 2007, 
2010). The Mann-Whitney test was used to compare two independent samples, Kruskall- 
Wallis for k comparisons, Spearman correlation rank test to quantify associations between 
two variables. Statistics were performed using R. (Ihaka and Gentleman 1996). 

3. Results 

3.1 General characteristics of sampled material 
3.1.1 Otters 

Otters have been systematically collected since the beginning of the toxicological program 
along Loire River and tributaries catchment (2004). This program allowed an increase in the 



298 



Pesticides in the Modern World - Risks and Benefits 



scientific use of previously collected and stocked individuals, for toxicological analyses first, 
but also for genetic study of otter recolonization, diet, biometry or causes of mortality 
approaches (Mucci et al. 2010). Main characteristics of otters analyzed in this study are 
summarized in table 2. 



Otter 


Sex 


Age 


Body 

index 

K 


Catchment 
Origin 


Cause of 
death 


Otter 


Sex 


Age 


Body 

index 

K 


Catchment 

Origin 


Cause of 
death 


LF01 


Female 


Juvenile 


1,21 


Upper part 


Collision 


LM123 


Male 


Subadult 


1,07 


Lower part 


Collision 


LM02 


Male 


Adult 


0,91 


Upper part 


Collision 


LM124 


Male 


Subadult 


1,00 


Lower part 


Collision 


LM05 


Male 


Adult 


1,08 


Upper part 


Collision 


LM125 


Male 


Adult 


1,04 


Lower part 


Collision 


LM09 


Male 


Subadult 


1,11 


Upper part 


Collision 


LM126 


Male 


Adult 


1,04 


Lower part 


Collision 


LM12 


Male 


Subadult 


0,71 


Upper part 


Collision 


LM127 


Male 


Subadult 


1,10 


Lower part 


Collision 


LM13 


Male 


Adult 


0,91 


Upper part 


Collision 


LM129 


Male 


Adult 


1,16 


Lower part 


Collision 


LM14 


Male 


Adult 


1,07 


Upper part 


Collision 


LM130 


Male 


Juvenile 


1,17 


Lower part 


Collision 


LM16 


Male 


Juvenile 


0,62 


Upper part 


Natural 


LM141 


Male 


Juvenile 


0,85 


Lower part 


Collision 


YL 


Male 


Juvenile 


0,82 


Upper part 


Starvation 


LM144 


Male 


Adult 


0,89 


Lower part 


Collision 


LF62 


Female 


Juvenile 


0,95 


Lower part 


Collision 


LM148 


Male 


Adult 


1,20 


Lower part 


Collision 


LF64 


Female 


Adult 


1,01 


Lower part 


Collision 


LM153 


Male 


Adult 


1,36 


Lower part 


Collision 


LF68 


Female 


Juvenile 


0,64 


Lower part 


Collision 


LM154 


Male 


Subadult 


1,17 


Lower part 


Collision 


LM71 


Male 


Adult 


1,28 


Lower part 


Collision 


LM156 


Male 


Juvenile 


1,12 


Lower part 


Collision 


LF72 


Female 


Adult 


1,06 


Lower part 


Collision 


LF157 


Female 


Subadult 


0,82 


Lower part 


Collision 


LF74 


Female 


Subadult 


0,97 


Lower part 


Collision 


LF158 


Female 


Subadult 


0,58 


Lower part 


Collision 


LF 77 


Female 


Old 


0,67 


Lower part 


Collision 


LF159 


Female 


Juvenile 


1,03 


Lower part 


Collision 


LF78 


Female 


Adult 


0,71 


Lower part 


Collision 


LM160 


Male 


Adult 


1,20 


Lower part 


Collision 


LM83 


Male 


Adult 


0,68 


Lower part 


Collision 


LF161 


Female 


Adult 


0,95 


Lower part 


Collision 


LF85 


Female 


Subadult 


0,96 


Lower part 


Collision 


LF162 


Female 


Adult 


0,98 


Lower part 


Collision 


LM86 


Male 


Adult 


1,39 


Lower part 


Collision 


LF163 


Female 


Adult 


0,83 


Lower part 


Collision 


LF88 


Female 


Adult 


1,02 


Lower part 


Collision 


LF164 


Female 


Adult 


0,91 


Lower part 


Collision 


LF89 


Female 


Subadult 


0,96 


Lower part 


Collision 


LF165 


Female 


Adult 


0,95 


Lower part 


Collision 


LM90 


Male 


Adult 


0,89 


Lower part 


Collision 


LF167 


Female 


Adult 


0,92 


Lower part 


Collision 


LF91 


Female 


Adult 


1,03 


Lower part 


Collision 


LF168 


Female 


Adult 


1,06 


Lower part 


Collision 


LF93 


Female 


Adult 


0,82 


Lower part 


Collision 


LF169 


Female 


Adult 


1,05 


Lower part 


Collision 










LM94 


Male 


Adult 


1,38 


Lower part 


Collision 









Table 2. Main characteristics and causes of death of otters in this study. 

Fifty-one otters were necropsied and analyzed for this study, with 24 females (47%) and 27 
males (53%). Sex-ratio of the sample was very close to the equilibrium but characterized by a 
slight over-representation of males. This was noted in previous studies, and was generally 



Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic 

Food Webs: The Case of Eurasian Otter (Lutra lutra) and Osprey (Pandion haliaetus) in Loire ... 299 

attributed to the larger territory of males compared to females, with associated higher risk of 
vehicular collision during food or new habitat foraging, particularly in the case of natural 
recolonization of unknown habitats (Foster-Turley et al. 1990; Rosoux and Tournebize, 1993; 
Kruuk, 2006; Lemarchand, 2007). Most of dead otters (30 out of 51, i.e. 59%) were adults, 11 
were subadults, 9 were juvenile and only one was an old individual. This mortality picture 
is different from those observed in previous studies, where most of discovered otters were 
juvenile or subadults, with an linear increasing in probability of death with age, considering 
the rareness of very old individuals in nature (Kruuk and Conroy, 1991; Kruuk, 2006; 
Lemarchand, 2007). 

With the exception of only two individuals found in the wild during the study, all otters 
died after a vehicular collision. These results tend to confirm that road casualties seem to be 
one of the main causes of mortality of otters, but, as suggested by Kruuk and Conroy (1991), 
and Kruuk (2006), it is the easiest way to find dead otters, those dying of other causes in the 
wild having far less probability of being found. This bias of carcasses collect was difficult to 
overlap, because of the huge human and financial costs of systematic search on riverbanks, 
ponds and lakes in such a study area. Considering this bias of collect, assessing the real 
hierarchy of causes of mortality remains hard for such a species. Among those found in the 
wild, one was an adult found dead without any clinical sign or injury, the other was a very 
little otter, only a few days aged, died of starvation after the dead or the abandon by its 
mother. This finding was surprising: as young otters live all their time in den, the 
probabilities of discover them if they die is very low (Kruuk, 2006). With the exception of 
various injuries caused by road collisions, all otters were in good physical conditions, with 
no apparent organ damage due to intoxication, like hemorrhages, organ abnormality or 
wound. Body condition index was systematically comprised between 0,5 and 1,4: it can be 
assumed that none otter collected in this study was in poor health condition or particularly 
fat (index < 0,5 or > 1,4, respectively; Kruuk, 2006). Medium body condition index of all 
otters was 0,99, very close to the value of reference (=1; Kruuk, 2006). Differences between 
body condition indexes K, total length or weight of otters from upper or lower part of Loire 
River catchment were not significant. Post-mortem examinations of otters never showed any 
clinical sign of severe intoxication, like organ or tissue abnormality, secretions, hemorrhages 
or anemia. Lead pellets were found on two occasions in carcasses but were not a death 
causal agent. According to Bo Madsen et al. (2000) or Simpson et al. (2005), otters are 
generally few concerned by natural intoxication (e.g. botulism), viral or bacterial diseases. 
Individuals examined here never showed any strong disease or natural intoxication signs. 

3.1.2 Ospreys 

Osprey population in mainland France is monitored since the natural come back of the 
species as breeding one in 1984 (synthesised in Nadal and Tariel, 2008). From only one in 
1984, population of breeding ospreys in the study area increased to 35 active nests in 2010, 
the overall breeding success during 1985-2006 periods was 2.0 fledglings per active nest. 
This value is higher than the stable population threshold (=0,8), and, associated with the 
recorded survival rate of adults of 0.97, suggests a very good reproduction dynamics of 
osprey in the study area during this period (Poole, 1989; Rattner et al. 2004; Wahl and Tariel, 
2006; Dennis, 2008; Nadal and Tariel, 2008). The large, favourable and non-fully occupied 
potential habitats along the Loire River, associated with an important and diversified food 
resource were the main factors of this reproductive success. Ospreys have been 



300 



Pesticides in the Modern World - Risks and Benefits 



systematically collected in Mainland France from the end of 2007, when toxicological 
program on otter was extended to this top-predator. Main characteristics of ospreys 
analyzed in this study are listed in table 3. 

17 osprey samples collected since 2007 in France were used. As some of the birds were ringed, 
or came from known nests, information about age and origin was established for 12 ospreys 
(70%). 7 osprey samples (3 non hatched eggs, 3 dead pulli in nests and one adult) came from 
the breeding population along Loire River. The other birds were subadults or adults collected 
during spring or autumn migration: 4 from Germany, 1 from Norway, and 5 non-ringed birds 
were from unknown origin. 3 ospreys died after electrocution on power cables, 3 after illegal 
shots in spite of the full protection of the species by law. Drawing of ospreys in fishponds with 
inadequate protection nets is another cause of osprey mortality currently emerging: 4 
individuals died in the same structure during spring 2009 (March 23 rd , 24 th , 26 th and 28 th ) in 
eastern France. To minimize this drawing risk, protection nets were recently modified in 
several fishponds situated along osprey migration corridors. As observed concerning otters, 
post-mortem examination never showed any showed any clinical sign of severe intoxication, 
like organ or tissue abnormality, secretions, hemorrhages or anemia. Three pulli from the same 
nest died of starvation during a long period of bad weather conditions. Three cases of feather 
pitching syndrome were observed, but these specimen were not collected early enough to 
support toxicological analyses. The rest of the examined individuals were in good physical 
conditions (normal size and weigh) and did not show any intoxication or disease sign. 



Osprey 


Sex 


Age 


Origin 


Cause of death 


bbz 4 


female 


adult 


Germany 


electrocution 


Bbz 3 


male 


adult 


Loire River 


electrocution 


Bbz7 


unknown 


e gg 


Loire River 


non hatched egg 


Bbz8 


unknown 


e gg 


Loire River 


non hatched egg 


Bbz 9-11 


male 


juvenile 


Loire River 


pullus dead in nest 


Bbzl2 


unknown 


juvenile 


Loire River 


pullus dead in nest 


Bbz 13 


unknown 


e gg 


Loire River 


non hatched egg 


Bbz 14 


female 


subadult 


Norway 


illegal shot 


Bbz 17 


male 


subadult 


unknown 


electrocution 


Bbz 19 


male 


juvenile 


Loire River 


pullus dead in nest 


Bbz 20 


female 


adult 


unknown 


illegal shot 


Bbz 21 


male 


subadult 


Germany 


illegal shot 


Bbz 23 


female 


adult 


Germany 


drawn in fish farm 


Bbz 24 


female 


subadult 


unknown 


drawn in fish farm 


Bbz 25 


male 


subadult 


unknown 


drawn in fish farm 


Bbz 28 


male 


subadult 


Germany 


dead in health center 


Bbz 31 


male 


adult 


unknown 


drawn in fish farm 



Table 3. Ospreys' characteristics and causes of death analyzed in this study. Osprey 
numbers corresponds to the chronological sampling order. 



Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic 
Food Webs: The Case of Eurasian Otter (Lutra lutra) and Osprey (Pandion haliaetus) in Loire . 



301 



3.2 Contamination by organochlorine pesticides 

Results concerning contamination of otters by OC pesticides are represented in table 4. OC 
pesticides and especially DDT metabolites were detected in all (100%) of the analyzed otters, 
confirming the widespread exposure of otter habitat in France to OC pesticides (Colas et al. 
2006; Lemarchand et al. 2007, 2010). Mean concentrations of total OC pesticides in otter liver 
of the whole Loire River catchment reached 2,2 mg.kg- 1 lipid weight, without any statistical 
variations with the geographical origin of the individuals: the increase in concentrations by 
going downstream observed in the upper part of the catchment (Lemarchand et al. 2007) 
was not significant at the whole catchment scale. Differences of various OC pesticides with 
otter age or sex were not significant. DDT was detected in 17 individuals (33%), confirming 
quite recent uses of this insecticide, banned in 1973 in France. 15 of these 17 DDT- 
contaminated otters were coming from the lower part of Loire River catchment. However, 
DDE was the most abundant of the analyzed DDTs metabolites, confirming the general 
decrease of otter's exposure to DDT and OC compounds in Europe (Mason, 1998). Lindane 
constituted the most abundant OC pesticide after DDTs, with quite low concentrations. 
Aldrin, Dieldrin, Heptachlor and Heptachlor epoxide were very low, often close to the 
detection limits. Methoxychlor and Endosulfan were never detected in otters. Measured OC 
pesticides concentrations remained below the available thresholds concerning otter survival 
(Mason and Macdonald 1993 a,b; 1994). Considering the actual population dynamic in 
France and elsewhere in Europe, OC compounds are not supposed to constitute an 
immediate threat to otter conservation. 





Otters (n= 51) 


Ospreys (n- 17) 


OC pesticides 


Males 


Females 


Juveniles 


Sub 
adults 


Adults & 
Old 


Males 


Females 


Eggs 


Juveniles 


Sub 
adults 


Adults 


DDT 


0,02 


0,01 


- 


0,01 


0,02 


- 


- 


- 


- 


- 


- 


DDE 


1,12 


1,85 


0,1 


1,45 


2,01 


10,7 


0,55 


3,40 


- 


- 


1,86 


DDD 


0,54 


0,35 


- 


0,41 


0,60 


- 


- 


- 


- 


- 


- 


Lindane 


0,11 


0,08 


0,05 


0,15 


0,12 


- 


- 


- 


- 


- 


- 


Methoxychlor 


- 


- 


- 


- 


- 






- 


- 


0,01 


0,3 


Aldrin 


0,05 


0,04 


- 


0,04 


0,11 


- 


- 


- 


- 


- 


- 


Heptachlor 


0,01 


0,01 


0,01 


0,18 


0,14 


- 


- 


- 


- 


- 


- 


Hepta. epox. 


0,01 


0,01 


0,01 


0,01 


0,02 


- 


- 


- 


- 


- 


- 


Endosulfan 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 



Table 4. Contamination of otters and ospreys from the Loire River catchment by 
organochlorine pesticides (mg.kg- 1 ). 

Results concerning contamination of ospreys by OC pesticides are presented in table 4. OC 
pesticides were detected in all but 5 of the sampled ospreys, and maximum Z OC pesticides 
concentrations in liver reached 10,7 mg.kg- 1 lipid weight. Only DDTs residues (mainly p,p'- 
DDE) and Methoxychlor were found in samples. DDT by itself was never found in ospreys. 
These two compounds were never found simultaneously in the same samples. Lindane, 
Aldrin, Heptachlor, Heptachlor epoxide and Endosulfan were never found in samples. 
Endosulfan is the only OC pesticide never found in otter or osprey samples. Nevertheless, 
these compounds were noted in previous studies concerning ospreys, particularly 
concerning Lindane, Aldrin and Heptachlor epoxide (Ewins et al. 1999; Henny et al. 2003, 



302 Pesticides in the Modern World - Risks and Benefits 

2008; Toschik et al. 2005). This difference could be related to a different exposure of 
American ospreys to OC pesticides when compared to European ones, resulting in a higher 
OC pesticides accumulation pattern in the whole American population. Indeed, American 
ospreys were exposed to OC pesticides without interruption from the beginning of 
industrial uses until legal ban. In France, DDT and other OC pesticides were banned before 
the return of the osprey or at the beginning of population expanding, resulting in a lower 
and decreasing exposure to contaminants. DDE was detected in 4 individuals (24 %), 
including 2 eggs from 2 different nests along Loire River and 2 adults, one coming from 
Loire River. We did not observe any significant variations in OC pesticides concentrations 
with osprey age, sex or origin. DDE concentrations remained quite low (range 0.0 - 10,7 
mg.kg- 1 lipid weight). These values were comparable to those noted by Rattner et al. (2004) 
or Henny et al. (2008), and should not be of concern for osprey direct conservation. DDE 
concentration in available osprey eggs (n=3) reached 0.0, 4,6 and 5,9 mg.kg- 1 lipid weight, 
respectively. Concerning the latter, the measured values were slightly higher than the 4.2 
mg.kg- 1 (measured in wet weight) eggshell thinning threshold cited in the literature 
(Wiemeyer et al. 1988; Henny et al. 2008), but these eggs did not show any shell breakage and 
were not damaged. Methoxychlor was detected in 8 individuals (47%), with low values 
(range 0.0 - 0.93 mg.kg- 1 lipid weight, see table 1). General Methoxychlor mean reached 0.01 
mg.kg- 1 ww, far less than noted by Weber et al. (2003) in Germany, where 100% of the 
sampled ospreys were contaminated by Methoxychlor. Following these authors, we assume 
this compound is not a direct threat to ospreys. 

3.3 Contamination by organophosphate, carbamate and pyrethroids pesticides 

To a general point of view, contamination of otters and ospreys by the 16 highly toxic 
cholinesterase inhibitors appeared low and scattered, with only few individuals 
concerned. Only two otters (4%) and 8 ospreys (47%) were characterized by detectable 
cholinesterase inhibitors concentrations. Among the OP pesticides analyzed, 7 
(Mevinphos, Phorate, Malathion, Parathion, Methidathion, Disulfoton sulfone and 
Triazophos) were quantified in ospreys and are presented in table 5. Only two OP 
pesticides (Parathion and Methidathion) were detected in otters, with low concentrations 
(between 0,02 and 0,03 mg.kg- 1 ww, data not shown). No statistical comparison could 
have been made concerning otter contamination by OP pesticides. Other OP pesticides 
analyzed in otter and osprey tissues were never been detected. Carbamates pesticides 
(Methiocarb and Carbofuran) were not quantified in otters and ospreys during this study, 
however they were recently noted in intoxicated red kites (Milvus milvus) in France (Berny 
and Gaillet, 2008). It can be assumed that diet of otters and ospreys (based on fish) is less 
exposed to carbamates pesticides accumulation that other diet types of some terrestrial 
predators like red kite. 

OP pesticides were only measured in subadult and adult ospreys, and never found in eggs 
or pulli during this study. None of the individuals coming from the nesting population 
showed any OP pesticides contamination. OP pesticides variations with osprey age, sex or 
origin were not significant. Triazophos, Disulfoton sulfone and Mevinphos were the most 
frequently detected compounds in ospreys (n=4, 3 and 3, respectively). Phorate and 
Malathion were detected in only one individual, characterized by the highest diversity of 
compounds (4 compounds with also Parathion and Methidathion) and by the highest 
concentrations of total OP pesticides (0,9 mg.kg- 1 ww, see table 5). 



Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic 
Food Webs: The Case of Eurasian Otter (Lutra lutra) and Osprey (Pandion haliaetus) in Loire 




303 




Individuals 


bbz 
19 


bbz 

3 


bbz 

7 


bbz 
8 


bbz 
9-11 


bbz 

12 


bbz 
13 


bbz 
14 


bbz 

28 


bbz 

21 


bbz 

23 


bbz 

4 


bbz 
20 


bbz 

24 


bbz 

25 


bbz 

17 


bbz 
31 


Mevinphos 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


0,03 


- 


0,05 


- 


0,3 


- 


- 


Phorate 
































0,02 


- 


Malathion 
































0,03 


- 


Parathion 


- 


- 


- 


- 


- 


- 


- 


0,4 


- 


- 


- 


- 


- 


- 


- 


0,8 


- 


Methidathion 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


0,02 


- 


- 


0,02 


- 


Disulfoton 
sulfone 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


0,3 


- 


0,3 


- 


0,04 


Triazophos 
















0,02 


0,02 


- 


- 


- 


- 


0,03 


0,03 


- 


- 



Table 5. Contamination of ospreys by OP and CA pesticides (mg.kg- 1 wet weight). Data are 
organized according to geographic origin of individuals for a better comparison. 

As described above, ospreys were generally in good physical conditions (adequate mass and 
total body fat) and did not show any OP pesticides poisoning sign (e.g. diarrhea, pulmonary 
oedema, tightened claws, Berny and Gaillet, 2008) during post-mortem examination. 
Furthermore, some of them were collected during migration flows, and none bird was 
found with apparent sign of exhaust potentially brought about by contamination 
consequences. Measured concentrations remained well below toxic doses of cholinesterase 
inhibitors (documented as about 10 mg.kg- 1 ww) and were not death causal agent of these 
individuals. Low level of concentrations and of contamination cases frequency should not 
constitute a threat to the population level, taking into account recent restrictions on OP and 
CA pesticides uses. 

Pyrethroids pesticides residues were never found in any otter or osprey samples (data not 
shown). The good quality and abundance of samples and the efficiency of the method used 
avoided methodological bias in pyrethroids pesticides detection. 
These results lead to several hypotheses: 

Pyrethroids pesticides may be quickly degraded or metabolized by animals; 

Pyrethroids pesticides may be little concerned by bioaccumulation in aquatic food 

chains; 

Global accumulation and transfer of recently used pyrethroids is very low for the 

moment, but is able to raise in the future with increasing uses. 
Metabolite of pyrethroids (3-phenoxybenzoic acid 3-PBA) was investigated in osprey eggs 
of the Washington State, USA (Chu et al. 2007) without being found. Complementary 
studies are needed to precisely evaluate general contamination of fauna by pyrethroids 
pesticides. Insect-consumers birds in treated areas (e.g. Eurasian skylark Alauda arvensis, 
common quail Cotumix c.) and their bird-eating predators (e.g. Montagu's harrier Circus 
pygargus or western marsh harrier C. aeruginosus) could be used as sentinels for an evaluation 
of direct transfer of pyrethroids through terrestrial systems first, before generalization of 
analyses to other systems, like aquatic systems and associate predators. 



3.4 Contamination by herbicides 

As observed for OP and CA pesticides, contamination of otters and ospreys by the 8 
analyzed herbicides was generally low and few diversified. Only two otters (4%) and 7 
ospreys (41%) showed detectable herbicides concentrations. Of the 10 herbicides analyzed, 
Metholachlor was the only herbicide detected in otters, on two occasions and with low 
concentrations (0,02 and 0,05 mg.kg- 1 ww respectively, data not shown). Two herbicides 



304 



Pesticides in the Modern World - Risks and Benefits 



(Terbuthylazine and Alachlor for 5 individuals each) and fungicide Epoxyconazole (in only 
one case) were quantified in ospreys (see table 6). None of the ospreys from the nesting 
population in France showed any herbicide or fungicide contamination. Herbicides 
variations with osprey age, sex or origin were not significant. Herbicides were not found in 
osprey eggs during this study. It can be underlined a unique case of contamination by 
fungicide Epoxyconazole (5,64 mg.kg-1 ww, see table 6), detected in an osprey from 
Germany. This individual did not show any particular intoxication sign. As for OP and CA 
pesticides, concentrations of herbicides measured in tissues and low frequency of herbicide 
detection leads to a probable weak impact of these compounds on species' conservation. 
Nevertheless, herbicides were very rarely searched in ospreys and very few data are 
available in literature for comparison. Chu et al. (2007) reported contamination of osprey 
eggs by a Dacthal structural isomer, indicating that some herbicides could be accumulated 
in ospreys with a potential reproductive impact on populations. 



Individuals 


bbz 

19 


bbz 

3 


bbz 

7 


bbz 

8 


bbz 
9-11 


bbz 

12 


bbz 
13 


bbz 

14 


bbz 

28 


bbz 

21 


bbz 

23 


bbz 

4 


bbz 

20 


bbz 

24 


bbz 

25 


bbz 

17 


bbz 
31 


Terbuthylazine 
















0,09 


0,83 


0,3 


- 


- 


- 


0,54 


- 


0,88 


- 


Alachlor 


- 


- 


- 


- 


- 


- 


- 


0,01 


- 


0,01 


- 


- 


0,01 


0,08 


- 


0,04 


- 


Epoxyconazole 


- 


- 


- 


- 


- 


- 


- 


- 


- 


- 


5,64 


- 


- 


- 


- 


- 


- 



Table 6. Contamination of ospreys by herbicides and fungicides (mg.kg- 1 wet weight). 

4. Conclusion 

A large non-invasive program allowed an important sampling of European otter and 
osprey tissues for various pesticides contamination study. Results showed that otter and 
osprey could be used as good sentinels of organochlorine and, to a lesser extent, 
organophosphate pesticides and some herbicides accumulation in aquatic food chains. 
Carbamates and pyrethroids pesticides were not detected in those top-predators fish- 
eating species. Organochlorine, organophosphate pesticides and herbicides concentrations 
remained low and under values of concern for species direct short-term conservation. 
Regular increase in populations observed since three decades in France seemed to confirm 
a low impact of global contamination on otter and osprey. Nevertheless, long-term 
consequences of global contamination on otter and osprey behaviour (e.g. prey or habitat 
foraging, hunting, mating or territory defence), synergies or antagonisms between 
compounds or potential long-term endocrine disruptors effects of low-concentrated 
contaminants remains unknown and should be elucidated during future standard 
monitoring of these sentinels species. 



5. Acknowledgments 

Authors wish to thank the following structures or people for otter and/ or osprey 
conservation and study or samples providing (see text): Museum d' Orleans (sampling 
coordination), MEEDDM, DREAL Centre, LPO, UFCS, CRBPO, ONCFS, ONEMA, ONF, 
RTE, EDF, R. Wahl, private land owners and companies, Ph. Guillet & M.-F. Larigauderie of 
the City of Orleans Museum, D. Vey & the technical team of VetAgro Sup Toxicology unit, 
"Loiret Nature Environnement" and other environmental associations. 



Semi Aquatic Top-Predators as Sentinels of Diversity and Dynamics of Pesticides in Aquatic 

Food Webs: The Case of Eurasian Otter (Lutra lutra) and Osprey (Pandion haliaetus) in Loire ... 305 

This study was financially supported by: European Commission (FEDER), « Plan Loire 
Grandeur Nature 2007-2013 », Etablissement Public Loire, Agence de l'Eau Loire-Bretagne, 
French Ministry of Environment (MEEDDM, DREAL Centre, Plan National d' Actions pour 
le balbuzard pecheur en France), VetAgro Sup, Ville d' Orleans, Office National des Forets, 
Pare naturel regional des Volcans d'Auvergne, Pare interregional du Marais poitevin, and 
CNRS. 

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17 



Is Pesticide Use Sustainable in Lowland Rice 

Intensification in West Africa? 

F. E. Nwilene 1 et al.* 

^Africa Rice Center (AfricaRice), 

3 Ebonyi State University, 

4 National Cereals Research Institute (NCRI), 

international Consultant, 

^Institute of Agricultural Research for Development (IRAD), 

^Nigeria 

2 Benin 

5 England 

^Cameroon 



1. Introduction 

Rice is a major staple food for about 3 billion people (Nguyen and Ferrero 2006). In West 
Africa, it is indeed no longer a luxury food and has become a major source of calories for the 
urban poor. The poorest urban households obtain 33% of their cereal-based calories from 
rice (NISER, 2005). Urbanization, changes in employment patterns, income levels, and rapid 
population growth have contributed to widening the gap between supply and demand 
(Figure 1). The gap between production and consumption is made up by imports, which are 
estimated at 2 million metric tones per annum. 

Population, In billions 




:^:: :?^ r^; :--i-i ■-■-?:■ r; ; 75 .-k :~^ i^»y.---:i ::.y: xonzs :::^:::0 2C25zu30 2Q35 2:hd;u-5 2050 



Fig. 1. World Population Growth 1950-2050 (Source: United Nations, 2009). 



*A. Togola 2 , O. E. Oyetunji 1 , A. Onasanya 2 , G. Akinwale 1 , E. Ogah 3 , E. Abo 4 , M. Ukwungwu 4 , 
A. Youdeowei 5 and N. Woin 6 



312 Pesticides in the Modern World - Risks and Benefits 

Rice remains one major crop in which West Africa can easily become self-sufficient given the 
potentials that abound in the region. The potential land area for rice production in West 
Africa is between 4.6 million and 4.9 million ha. Out of this, only about 3.7 million ha — or 75 
percent of the available land area— is presently cropped for rice. Cultivable land to rice is 
spread over five ecologies, namely: rainfed upland, rainfed lowland or shallow swamp, 
irrigated rice, deepwater or floating rice and tidal mangrove swamp. The commonly used 
ecosystems and share of rice area for the rice ecosystems are presented in Table 1. 



Ecology 




Share (%) 




Yield 


(l/ha) 




Area 


Production 


Current 




Potential 


Upland 


40 


37 


1.0 




1.5-4.5 


Rainfed lowland 


48 


49 


1.4 




2.5-5.0 


Irrigated lowland 


6 


14 


2.8 




5.0-7.0 



Upland: area expansion and yield increase may be fulfilled 

Rainfed lowland: most promising 

Irrigated lowland: dam construction is too expensive at current rice price 

Source: Sakurai, 2006 

Table 1. Rice production ecology in West Africa 

Amongst these, lowland rice has the highest priority, being the ecology that represents the 
largest share of rice area and rice production. Smallholder farmers with farm holdings of 
less than 1 ha cultivate most of the rice produced in West Africa. However, rice productivity 
and production at the farm level are constrained by several factors. These constraints 
include insufficient appropriate technologies, poor supply of inputs, ineffective farmer 
organizations and groups, poor quality of rice, poor marketing arrangements, inconsistent 
agricultural input and rice trade policies, and environmental constraints. These 
environmental constraints include poor drainage and iron toxicity in undeveloped lowland 
swamps, poor maintenance of developed lowland swamps, drought, deficiencies of N and 
P, poor soil management practices, seasonal over-flooding of rice fields, pests and diseases. 

1.1 Hypothetical shift in production system 

With the increasing awareness of the limited potential for intensification of rice production in 
the uplands, farmers are gradually moving into the lowlands, which are less fragile (permits 
residual moisture use), more fertile and ecologically robust. The lowland areas are 
underutilized in West Africa. The lowland areas are expected to meet the growing demand for 
rice in West Africa because they provide potential for expansion, diversification and 
intensification of rice production in the region. This change in farming practice has been 
accompanied by an increase in the use of agrochemicals (pesticides and fertilisers), high- 
yielding varieties and monoculture/ continuous cropping, which further disrupt traditional 
farming and natural ecosystem functioning. Most West African countries are currently 
undergoing intensification in rice production to cope with the high population pressure. 
However, these may have adverse consequences on pest outbreaks, if it lacked the vision on 
the conservation of renewable inputs (biodiversity), and the whole issue of sustainability. This 
prediction is based on the hypothesis that with the improvement in the irrigation system, 
farmers will be able to grow high-yielding, photo-insensitive rice crops, and more crops per 
year on the same field. In such systems, the pest and beneficial cycles will be uninterrupted. 



Is Pesticide Use Sustainable in Lowland Rice Intensification in West Africa? 31 3 

1.2 Insect pests of rice in West Africa 

The major insect pests of lowland rice in West Africa include: the African rice gall midge 
(AfRGM), Orseolia oryzivora Harris and Gagne (Diptera: Cecidomyiidae); the rice stem borer 
complex: the stalk-eyed flies - Diopsis spp. (Diptera: Diopsidae); the African white borer - 
Maliarpha separatella Ragonot (Lepidoptera: Pyralidae); the yellow stem borers - Scirpophaga 
spp. (Lepidoptera: Pyralidae). Other important pests of rice include: vectors of rice yellow 
mottle virus (Trichispa sericea Guerin, Chaetocnema pulla Chapius, Chnootriba similis Thunberg 
and Oxya hyla Serville, etc.). All these pests are indigenous to West Africa except Maliarpha 
separatella that can also be found in Asia. 

1.3 Yield losses caused by insect pests 

Serious damage to the rice crop by the complex of insect pests result in significant yield 
losses which are typically in the range 10-30% yields and, in some regions or years, may 
exceed 90% (Nguu, 2008) (Table 2). Pests cause considerable and unacceptable crop losses in 
the field and in storage. The very high food losses in West Africa, attributable to pests, 
highlight their role in causing food shortages that lead to hunger. 

Country Pests Estimated crop loss 

Ghana 
Nigeria 

Burkina Faso 

Mali 
Cameroon 

Source: Youdeowei (1989, 2004) 

Table 2. Examples of average losses attributable to pests of rice in selected West African 
countries 

1.4 The race for pesticide sales in West Africa 

The yield potential of rice cultivated in the intensified systems is continually challenged by 
chronic pest infestations and by pest outbreaks. This challenge is seen by the chemical 
industries mostly based in Asia and developing countries as an avenue to aggressively 
market their products in West Africa. The products are sold without proper training of 
smallholder farmers on how to safely apply it and without warning of the harmful effect on 
the environment. The sad aspect of the race for pesticide sales is that many banned 
pesticides in Asia and other developed countries of the world are being dumped in West 
Africa. The indiscriminate use of the pesticides has posed a lot of danger to the environment 
and ecosystem making human life to be under threat. The current article, therefore, not only 
meets a demand expressed from the regional entomologists but also makes an important 
contribution to raise alarm of the danger of pesticides in lowland rice which is currently 
being targeted for intensification. 

2. Importation of pesticides into West Africa 

As agricultural production system moves more and more from subsistence to market - 
oriented large scale farming, a concomitant increase in pesticide usage arise (Sosan et al, 



Pests 


Estimated cro] 


Stem borers 


30% 


Stem borers 


25 - 30% 


African rice gall midge 


10-35% 


Stem borers 


10 - 40% 


African rice gall midge 


20 - 60% 


African rice gall midge 


20-35% 


Stem borers 


26 - 30% 



314 



Pesticides in the Modern World - Risks and Benefits 



2008). The climatic conditions of West Africa especially rainfed lowland ecology is 
conducive for build up of pest populations. Pesticide use in Africa accounts for less than 5% 
of global pesticide use and per hectare averages are low, estimated at around 1 kg/ha active 
ingredient applied (compared with 3- 7kg/ha in Latin America and Asia (PAN, 2010). 
However, low use volumes do not necessarily equate to low risk, particularly as some of the 
most toxic pesticides continue to be applied in Africa especially in West Africa, often under 
extremely dangerous conditions (PAN, 2010). Though, there are differences in the rate of 
agrochemicals application across the agroecological zones, pesticide use was high in dry 
savannah of West Africa (Ephraim et al, 2010). Pesticide use in Africa accounts for only 2- 
4% of the global pesticide market of US$31 billion (Williamson et al, 2008). Although Africa 
is currently neither a major consumer nor producer of chemicals in global terms, pesticides 
use in the African agricultural sector is likely to increase as a result of the growing 
commercialization as well as the growing focus of development agencies on improving 
yields of small farmers (Nelson et al, 2006). Most African countries were net importers of 
pesticides. In Ghana, the number of pesticides dumped by the chemical industries was 
between 163 to 180 units as at 2002 (Suglo, 2002). In Kabba area of Kogi State, Nigeria, the 
number of pesticide users increased dramatically from 42% in 1971 to 78% in 1998 (Youm et 
al., 1990). Importation of agrochemicals into sub-Sahara Africa increased in monetary values 
from $16.1 million in 1973 to $30 million in 1977 (Youm et al, 1990). Most of the pesticides 
brought into West African countries have been banned. Pesticide that is banned for 
agricultural purposes in 52 countries due to its hazardous nature is being used in Ghanaian 
agriculture (Glover et al, 2008). Most farmers in Africa increasingly depend on pesticides 
alone to control insect pest, and without satisfactory understanding of the associated 
hazards. Nigeria ranked first among West African countries in terms of quantities of 
pesticides use (Abete et al, 2000). Thus, Nigeria alone accounted for nearly 93% of UK 
pesticide exports to West African countries. Pesticides are the main sources of pollution in 




■k 



Fig. 2. Pesticide application on rice field 



Is Pesticide Use Sustainable in Lowland Rice Intensification in West Africa? 31 5 

the Senegal River Valley of Senegal, Mauritania, and Mali principally for vegetable production 
and herbicides/ fertilizers for irrigated rice cultivation. Overall, we do not want to experience 
pest and disease resurgence as a result of high use of chemical pesticides (Figure 2). The only 
way we can prevent it or reduce the negative effect is to educate irrigated rice farmers on the 
danger ahead of the indiscriminately use of pesticides. There is high overuse of chemical 
fertilizers and pesticides in cotton compared to rice in West Africa. 

3. Cases of pesticide mis-use 

Over the decades, chemical pesticide use has posed a threat to subsistence farming in West 
Africa because of the well known technical drawbacks such as high cost, lack of adequate 
protection for the user, absence of safety warnings, excessive and wasteful use leading to 
environmental pollution. A case in point is the Gezira irrigation scheme in Sudan, where 
continuous use of pesticides against the cotton jassid, Empoaca lybica has led to resistance in 
the whitefly (Bemisia tabaci), cotton bollworm (Helicoverpa armigera), and aphids (Aphis 
gossypii). This, in turn, has led to even higher rates of pesticide application and the 
consequent emergence of secondary pest outbreaks due to the selective removal of natural 
enemies from the crop system. For instance, citrus leafminer (Liriomyza trifolii) is native to 
Asia but has been a minor pest of citrus in Africa until recent years when it is now 
considered as the major threat to citrus (Abete et ah, 2000). The picture was not different in 
Madagascar where Spodoptera littoralis became a serious pest due to over-use of chlorinated 
hydrocarbons including monophos-DDT against cotton pests. Pesticide overuse to control 
pests in other crops such as cotton, coffee, cacao, groundnuts has resulted in the 
development of resistance to dieldrin and DDT by two mosquito species, Anopheles gambiae 
Giles and Anopheles rofipes (Gough) in the West African countries of Ivory Coast, Nigeria, 
Ghana, Mali, Burkina Faso, Togo, and Senegal. 

In South East Asia, Brown Planthopper (BPH), a secondary pest of rice, suddenly became a 
major pest due to insecticide misuse. Since 2005, outbreaks of rice BPH have occurred in 
East-Asian countries such as Vietnam, China and Japan. 

3.1 Destruction of non-target organisms and natural enemies 

Non-target organisms are organisms that the pesticides are not intended to kill. Natural 
enemies include insect predators, insect parasitoids, and insect pathogens. Over 98% of 
sprayed insecticides reach a destination other than their target species, including non- target 
species. Successful biological control using five exotic parasitoids against the potato tuber 
moth, Phthorimaea operculella, both native of South America was achieved in Zimbabwe and 
Zambia. Unfortunately, this system has broken down due to increase in pesticide use by 
farmers unaware of the value of biological control, and due to the need other pests. Overuse 
of pesticides in Ghana to control cocoa mirids resulted in the killing of numerous non-target 
beneficial organisms. As a consequence, the shield bug, Batllycoelia thalassina 
(HerrichSchaeffer), a secondary pest resurged and caused a yield loss of 18% of the cocoa 
crop in Ghana's Eastern and Brong-Ahafo Regions (Owusu, 1971, Alfred et al., 2001,). 

3.2 Human and animal health hazards 

Chemicals pollute the water body thus making it unsafe for human use e.g. drinking, 
washing of farm produce, etc. Many of the pesticides used are persistent soil contaminants, 



316 Pesticides in the Modern World - Risks and Benefits 

whose impact may endure for decades and adversely affect soil conservation (USEPA, 2007). 
Pesticide related poisoning deaths are often caused by using pesticide packages or 
containers after they are emptied of toxicants. It was reported by Youm et al. (1990) that 
forty six residents in Ilorin area of Nigeria were hospitalized as a result of "mistakenly 
drinking or eating pesticides". Also, in a study conducted by Hotton et al. (2010) in the 
northeastern part of Nigeria on effect of pesticide use, he found out various ailments 
associated with pesticide use and the use of pesticide container. These include: bronchilis 
chest pain, asthma, cough, running nose, vomiting, nausea, excessive sweating, diarrhea, 
burning on urination, abdominal pain, irritation of eye, temporarily and permanent lost of 
vision, weakness of arms, hands and legs, stiffeners of the waist, fatigue, etc. Empty 
pesticide containers are used to store food because of a lack of understanding on dangers of 
pesticides, poor pesticide labeling, and a low literacy rate. Pesticides that are applied to 
crops can volatilize and may be blown by winds into nearby areas, potentially posing a 
threat to wildlife (Sequoia & Kings, 2007). More importantly, the remains of these pesticides 
flow back to the streams and river. Some people at the other end will fetch it for drinking 
and for other domestic activities thus resulting to one ailment or the other depending on the 
concentration. Fish and other aquatic biota may be harmed by pesticide-contaminated water 
(Collin et al, 2008). 

4. Beyond pesticide application 

Resistance in pests due to chemical application is one of the major factors disrupting 
traditional pest management practices in West Africa. In order to maximize rice production 
and agricultural intensification while minimizing reliance on expensive pesticides, a long- 
term pest management strategies including varietal resistance, biological control and 
improved cultural practices is needed. IPM seeks to integrate multidisciplinary approach 
(combination of options) with limited pesticide use to provide effective environmentally 
sound, socially acceptable and economically safe solution to pest problems. AfricaRice and 
partners have developed some chemical free products for smallholder farmers in West 
Africa. Specific examples are provided below: 

4.1 Varietal resistance 

Improving varietal resistance or tolerance to insect pests is one of the most promising 
options for managing insect pests in West Africa. AfricaRice and partners have identified 
several Oyrza sativa varieties with resistance/ tolerance to the AfRGM. Cisadane (from 
Indonesia) has been selected as a variety tolerant to AfRGM and released in Nigeria as 
FARO 51 based on initial selection at NCRI and on-farm studies in Abakaliki by AfricaRice. 
BW 348-1 (from Sri Lanka) has good tolerance to AfRGM and iron toxicity under field 
conditions. It has been released in Burkina Faso and Mali (WARDA, 2003). Leizhung (from 
South Korea) is another tolerant variety to AfRGM released in Mali. Suitable lowland 
NERICAs being screened for insect resistance or tolerance include: NERICA L-25 and 
NERICA L-49 (Nwilene et al., unpubl. data). AfricaRice identified one tropical O. sativa 
variety (TOS 14519 from The Gambia) with moderate resistance to AfRGM, which is 
currently used as a resistant check variety in screening. Several traditional Oryza glaberrimas 
(e.g. TOG 7106 - from Mali, TOG 7206 - from Cote d'lvoire, TOG 7442 - from Nigeria and 
TOG 6346 - from Liberia) have been found to be highly resistant to the pest (Nwilene et al., 
2002). 



Is Pesticide Use Sustainable in Lowland Rice Intensification in West Africa? 31 7 

4.2 Biological control 

Biological control is a major component of sustainable agricultural systems that are 
designed and managed to reduce dependence on chemical and other energy-based inputs, 
minimize ecological risk resulting from farming practices, and enhance agricultural 
productivity in relation to resources available. To ensure that biological control will 
contribute to sustainable agriculture, AfricaRice identified the gregarious endoparasitoid 
Platygaster diplosisae (Hymenoptera: Platygastridae) and the solitary ectoparasitoid 
Aprostocetus procerae (Hymenoptera: Eulophidae) are the most important wasps attacking 
AfRGM. The Paspalum gall midge (PGM) Orseolia bonzii Harris (Diptera: Cecidomyiidae) 
which infests Paspalum scrobiculatum L. (Poaceae), a common weed in rice agroecosystems, is 
distinct from AfRGM, and is an alternative host for the two main parasitoids of AfRGM. The 
delay between the destruction of Paspalum scrobiculatum and the appearance of AfRGM 
populations on a rice crop means that the large majority of the parasitoids from O. bonzii die 
before AfRGM population is available - asynchrony between pest and associated natural 
enemies. AfricaRice has shown that habitat manipulation with Paspalum scrobiculatum 
management at the edge of rice fields had significantly increased the carry-over of 
parasitoids from Paspalum gall midge (Orseolia bonzii) to AfRGM. The combination of 
beneficial organisms, tolerant varieties and habitat management suppressed AfRGM, 
restored nature's balance, and resulted in increased rice yields (Nwilene et al., 2008a). 

4.3 Chemical-free products 

Chemical free products for insect pest control include the use of botanicals and biological 
control using pathogens. AfricaRice has demonstrated that neem seed powder and neem oil 
can provide effective control against termites in West Africa (Nwilene et al., 2008b). 
Termites constitute a major biotic constraint to upland rice production in West Africa. The 
control of termites has largely relied on broad spectrum and persistent organochlorine 
insecticides. Land use practices can affect the flow of water and persistent pesticides along 
toposequences from the fragile upland to the lowlands thereby causing harmful effect to 
humans. To meet the needs of upland rice farmers in West Africa, AfricaRice has shown that 
the biological control pathogen - the entomopathogenic fungus Metarhizium anisopliae is 
effective against termites on rice fields and can also be used as alternative to persistent 
chemical pesticides because of the serious health and environmental risks in terms of 
pollution, destruction/ death of non-target/ useful insects, and the reduction of biodiversity. 

4.4 Adoption of IPM practices 

The Food and Agriculture Organization of the United Nations in collaboration with 
technical assistance from AfricaRice introduced the concept of IPM training in farmer field 
school (FFS) to West Africa through a series of technical cooperation projects in irrigated rice 
schemes in Ghana, Cote d'lvoire and Burkina Faso. Following the success of this 
programme, IPM farmer field school projects were extended to several other countries in 
West, Eastern and Southern Africa. The initial results obtained by farmers who applied IPM 
practices for irrigated rice production in Ghana showed that yields of rice were consistently 
higher in IPM fields than in fields where conventional farming practices were adopted. In 
the rice fields where farmers adopted IPM practices, pesticide use for pest control was 
reduced by over 90% and savings on pesticide use amounted to $100 per ha. Net returns 
from such fields were 32% higher than in farmer practice fields. Data from Mali show 



31 8 Pesticides in the Modern World - Risks and Benefits 

conclusively that by adopting IPM practices farmers are able to increase the production of 
rice by 9 - 21%, increase revenue by 14% to 35% while at the same time significantly 
reducing pesticide use by up to 100% (Nacro, 2000; Youdeowei, 2001 and 2004). 

5. Conclusion 

The need to increase rice yields for present and future generation requires that solutions to 
these pest problems are found that are both sustainable and adoptable in the socio-economic 
environment of farming communities. Why are there abundant and diverse natural enemies 
in West Africa rice ecosystems? The answer is simple - low use of pesticides in rice fields. 
The high cost of pesticides means that few farmers have access to them at present. 
AfricaRice has demonstrated that managing, rather than destroying, a "friendly weed" 
(Paspalum scrobiculatum) at the edge of rice fields (good sources of parasitoids - Platygaster 
diplosisae & Aprostocetus procerae close to the rice crop) offers African farmers free, non- 
chemical control of the continent's worst rice insect pest - African rice gall midge (Nwilene et 
ah, 2008b). It is significant to note that the natural enemy populations of rice pests are high 
and the species diverse in West African rice ecosystems. This is evident in the high levels of 
parasitism of the AfRGM. This may be the reason why West Africa has not suffered the 
insect resurgent crisis, that occur in Asian rice ecosystems, where the natural enemies of the 
brown planthopper, Nilaparvata lugens (Stal) are being killed through the misuse and 
inefficient application of insecticides. This provides evidence that pesticide use is less in 
West African rice ecosystems and that the natural enemies are being conserved. All future 
IPM strategies development should be designed to preserve, and possibly enhance, the 
existing and abundant natural enemy populations in West African rice ecosystems. Whereas 
the challenge in Asia is to stop farmers from overuse of pesticides, in West Africa, it is to 
prevent future overuse of pesticides. IPM is the only preventive approach and the way out 
for pest management in lowland rice ecology. In the long-term, everyone benefits from a 
healthier environment. This generation must rise up to the task of saving the global 
environment from pollution by discouraging production and importation of synthetic 
pesticides into West Africa. Smallholder farmers who use pesticides are often unaware of 
the adverse effect of pesticide applications. In implementing integrated pest management 
options, existing farmers' knowledge should be carefully analyzed, refined and integrated 
into the basket of options for them to choose from. There is a need to revisit a number of 
national policies related to food production and protection, in order to encourage 
partnership and participation in the identification, analysis, advocacy, and follow-up of 
plant protection issues as well as public awareness of the effect of pesticides on food and the 
environment. 

6. References 

Abete, T., A. V. Huis and J. K. Ampofo. 2000. Pest management strategies in traditional 
agriculture: An African perspective. Annual Review of Entomology 45: 631-659. 

Alfred G., J.V.Suglo, M. Braun. 2001. An Economic and Institutional Analysis of Current 
Practice and Factors Influencing Pesticide Use. Pesticide Policy Project Publication 
Series No. 10. pp 1-127. 



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Collin, A. E., T. H. Suchanek, A. E. Colwell, N. L. Anderson and P. B. Moyle. 2008. Changes 

in fish diets and food web mercury bioaccumulation induced by an invasive 

planktivorous fish. Ecological Applications 18(8): 213-226. 
Ephraim, N, J. Pender, E. Kato, O. Omobowale, D. Phillip, S. Ehui 2010. Nigeria Strategy 

Support Program. International Food Policy Research Institute (IFPRI) 9. ppl-4. 
Hotton A.J, J.T. Barminas, S.A Osemeahon, T. Aboki. 2010. Monitoring of cholinesterase 

inhibition among retailers of agrochemicals in Northeastern Nigeria. European 

Journal of Scientific Research 46(1): 028-035. ISBN1450-216X. 
Nacro S. 2000. La formation participative en gestion integree des depredateurs des cultures 

a travers les champs-ecoles des producteurs au Mali. Etudes et Recherches 

Saheliennes No. 4/5: 74-80. 
Nelson M. and J. Mohamed-Katerere 2006. Africa environment outlook: our environment, 

our wealth chemicals. UNEP 2: 250-374. 
Nguu V. N. 2008. Sustainable intensification of rice production for food security in the near 

future - A summary report by the secretary, International Rice Commission. Pp 1- 

14. 
NISER (Nigerian Institute of Social Economic Research). 2005. An Overview of the Nigerian 

Rice Economy: The Nigerian Institute of Social and Economic Research (NISER), 

Ibadan - Nigeria No. 32. pp. 9. 
Nwilene, F.E., C.T. Williams, M.N. Ukwungwu, D. Dakouo, S. Nacro, A. Hamadoun, S.I. 

Kamara, O. Okhidievbie, F.J. Abamu and A. Adam 2002. Reactions of differential 

rice genotypes to African rice gall midge in West Africa. International Journal of 

Pest Management 48(3), 195-201. 
Nwilene F. E., K.F. Nwanze and O. Okhidievbie. 2006. African rice gall midge: biology, 

ecology and control. Field Guide and Technical Manual. Africa Rice Center 

(AfricaRice), Cotonou. 20 pp. 
Nwilene, F.E., A.Togola, T.A. Agunbiade, O.E. Ogah, M.N. Ukwungwu, A. Hamadoun, S.I. 

Kamara, and D. Dakouo 2008a. Parasitoid biodiversity conservation for sustainable 

management of the African rice gall midge, Orseolia oryzivora (Diptera: 

Cecidomyiidae) in lowland rice. Biocontrol Science and Technology 18(10), 1075- 

1081. 
Nwilene, F.E., T.A Agunbiade, A. Togola, O. Youm, O. Ajayi, S.O. Oikeh, S. Ofodile and O. 

O. Falola 2008b. Efficacy of traditional practices and botanicals for the control of 

termites on rice at Ikenne, Nigeria. International Journal of Tropical Insect Science 

28(1), 37-44. 
Owusu, M. E. 1971. Bathycoelia tlwlassino, another serious pest of cocoa in Ghana. Coconut 

Marketing Board Newsletter (Accra, Ghana) 467: 12-14. 
PAN (Pesticide Action Network). 2010. Understanding the full costs of pesticides: 

Experience from the Field, with a Focus on Africa. Pesticide Action Network (PAN) 

UK No 2: 26-27. 
Sakurai T. 2006. Intensification of rainfed lowland rice production in West Africa: present 

status and potential green revolution. The Developing Economies 44(2): 232-251. 
Sosan, M.B., E.A. Amos, A.O. Isaac and A. D. Muheez. 2008. Insecticide residues in the 

blood serum and domestic water source Cacao farmers in Southwestern Nigeria. 

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320 Pesticides in the Modern World - Risks and Benefits 

Suglo J. V. 2002. Pesticides dealers' Handbook. Ministry of Food and Agriculture, Ghana. 

Plant Protection and Regulatory Services Directorate, P. O. Box 37, Accra Ghana, pp 

1-65. 
United Nations. 2009. Population Division of the Department of Economics and Social 

Affairs. World Population Prospects: The 2008 Revision. Pp. 1-9. 
USEPA (US Environmental Protection Agency). 2007. United Nations Population Division, 

World Population Prospects, Pesticide registration (PR): (www. epa.gov). 19 
WARD A (West Africa Rice Development Association). 2003. Cisadane - a gall midge 

tolerant variety released in Nigeria as FARO 51 in 1998. A flyer. 4 pp. 
Williamson S., A. Ball and J. Pretty. 2008. Trends in pesticide use and drivers for safer pest 

management in four African countries. Journal of Crop Protection 27 (10):1327- 

1334. 
Youm O., F. E. Gilstrap, and G. L. Teetes. 1990. Pesticides in traditional farming systems in 

West Africa. Journal of Agricultural Entomology 7(3): 171-181. 
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economic importance. In: Yaninek, J.S. and Herren, H.R. (eds.) Biological Control: a 

Sustainable Solution to Crop Pest Problems in Africa, IITA, Ibadan, Nigeria, pp. 31- 

50. 
Youdeowei, A. (2001) A review of the FAO/RAFA crop protection sub-programme. 

Consultancy Report to FAO/RAFA, Accra, Ghana. 
Youdeowei, A. 2004. Fighting Hunger and Poverty: IPM Contributions in Africa. Plenary 

Lecture at the International Plant Protection Congress, Beijing, China. 22 pp. 



18 



Transgenic Pesticidal Crops and the 
Environment: The Case of Bt Maize 

and Natural Enemies 

Dennis Ndolo Obonyo 1 and John B. Ochanda Ogola 2 

1 International Centre for Genetic Engineering and Biotechnology, 

2 University ofVenda, 
South Africa 



1. Introduction 

Stem borers are the most destructive field insect pests of maize (see Plate 1) in sub-Saharan 
Africa (SSA) (Insect Resistant Maize for Africa [IRMA], 2001; Songa et al., 2001). Important 
stem borer species include Busseola fusca Fuller (Lepidoptera: Noctuidae), Chilo partellus 
Swinhoe (Lepidoptera: Crambidae) and Sesamia calamistis Hampson (Lepidoptera: 
Noctuidae) (Overholt et al., 1994). 




Plate 1. Sesamia calamistis larvae feeding on a maize leaf 

Stem borer control approaches that have been used (with varied degrees of success) fall 
into four broad categories: chemical (application of insecticides); cultural (use of a range 
of farm practices to delay or reduce insect attack); biological (use of natural enemies of 
stem borers); and host plant resistance (the plant offers its own resistance to insects). The 
use of Bt maize (genetically modified maize expressing a 5-endotoxin from Bacillus 
thuringiensis and therefore having an in-built ability to produce pesticidal toxins) has been 
found to be effective in the management of stem borers in other parts of the world 
(Sharma & Rodomiro, 2000). However, this strategy has not been widely employed in 
Africa despite recent efforts to develop Bt maize suitable for different agro-ecological 
zones in the region (Muhammad & Underwood, 2004). Also, there is still significant 
debate regarding the possible risks posed by this technology (Obonyo et al., 2010). Fears 



322 Pesticides in the Modern World - Risks and Benefits 

that have been raised include; food safety and human health concerns, environmental 
concerns, possible impact on agricultural systems, and socio-economic issues. Regulatory 
decisions on whether or not to adopt genetically modified (GM) crops should therefore 
take all these concerns into consideration. 

Because the Bt toxin is embodied in the plant itself, Bt crops are regulated as pesticides in 
some jurisdictions. For example, the US Environmental Protection Agency (EPA) has for a 
long time regulated Bt crops under the Federal Insecticide, Fungicide, and Rodenticide 
Act (FIFRA) (Frisvold & Reeves, 2010). The Food and Agriculture Organization (FAO) 
defines a pesticide as "any substance or mixture of substances intended for preventing, 
destroying or controlling any pest, including vectors of human or animal disease, 
unwanted species of plants or animals causing harm during or otherwise interfering with 
the production, processing, storage, transport or marketing of food, agricultural 
commodities, wood and wood products or animal feedstuffs, or substances which may be 
administered to animals for the control of insects, arachnids or other pests in or on their 
bodies". The term includes substances intended for use as plant growth regulators, 
defoliants, desiccants, agents for thinning fruit or preventing the premature fall of fruit, or 
any substances applied to crops either before or after harvest to protect crop produce 
from deterioration during storage and transport (Food and Agriculture Organization of 
the United Nations [FAO], 2002). 

Used within the context of Integrated Pest Management (IPM), Bt crops offer a number of 
advantages. They are safe and easy to use, requiring only planting seeds of an adapted, 
resistant cultivar (Kennedy, 2008). In general, resistant cultivars have been compatible with 
other IPM tactics, including cultural, biological and chemical controls (Smith, 2005, as cited 
in Kennedy, 2008). However, it is well established that plant-borne factors that affect 
herbivores also interact with natural enemies and consequently with the biological control 
function they provide. Natural enemies such as predators and parasitoids fulfil an 
important ecological and economic function by helping to keep stem borer populations 
below the economic injury level and thus contributing to sustainable IPM systems (Romeis 
et al., 2008a). Most IPM systems aim to enhance biological control through conservation of 
existing natural enemies (Bale et. al., 2008 as cited in Romeis et al., 2008a). Thus it is 
important to minimize the non-target effects of other components of IPM such as pesticides 
or habitat manipulation (Romeis et al., 2008a). 

Transgenic insecticidal plants can have impacts on natural enemies (Kennedy & Gould, 
2007, as cited in Romeis et al., 2008a); this may stem from changes in either the plant 
structure, or primary/ secondary metabolites. Adverse effects may occur, for example, if the 
natural enemy is exposed to, and is susceptible to the plant-borne insecticidal factor. These 
factors can cause population level effects which might lead to changes in the level of 
biological control that natural enemies provide (Kennedy & Gould, 2007, as cited in Romeis 
et al., 2008a). This chapter reviews published literature on impacts of Bt maize on stem borer 
natural enemies, with particular attention to stem borer parasitoids. This is aimed at 
consolidating information pertaining to the potential impacts of Bt maize on the 
development and behaviour of maize stem borers and their natural enemies, with special 
emphasis on stem borer parasitoids such as the larval parasitoids Cotesia flavipes Cameron 
(Hymenoptera: Braconidae), Cotesia sesamiae Cameron (Hymenoptera: Braconidae) and 
Xanthopimpla stemmmator Thunberg (Hymenoptera: Ichneumonidae). However, this is not 



Transgenic Pesticidal Crops and the Environment: The Case of Bt Maize and Natural Enemies 323 

an environmental risk assessment of Bt maize but an analysis of the possible impacts of Bt 
maize on one category of beneficial organisms in the ecosystem. 

2. Bt maize 

The Bt maize plant has a built-in system that consistently delivers the Bt toxins to the target 
pest throughout the growing season. Bt maize has been used to control a common maize 
stem borer, Ostrinia nubilalis Htibner (Lepidoptera: Pyralidae), in the northern temperate 
region (Matilde et al v 2006). Indeed, Bt maize has been commercially grown in the U.S.A 
since 1995 and the area under its cultivation is increasing (Minorsky, 2001; Sakiko, 2006). In 
Africa, Bt maize is commercially grown only in South Africa, though its cultivation is 
expected to spread to other countries of SSA (James, 2001). 

Bacillus thuringiensis toxins expressed in current commercially available Bt maize hybrids are 
selective in their mode of action (Swadener, 1994). Therefore some authors (e.g., Schuler et 
al, 1999a) claim that the effects of Bt maize on non-target arthropods associated with maize 
should be minimal. However, it was Bt maize that was involved in significant controversy 
(the "monarch butterfly controversy"), following the publication, in Nature, of a preliminary 
study by Losey et al. (1999) (Minorsky 2001). Indeed, Losey et al. (1999) raised serious 
concerns about the ecological safety of Bt maize cultivation to non-target lepidopterans, in 
particular the larvae of the monarch butterfly, Danaus plexipus L. (Lepidoptera: Danainae). 
On the basis of laboratory assays, the authors concluded that monarch larvae reared on 
milkweed (Asclepias syriaca) leaves dusted with pollen from Bt maize ate less, grew more 
slowly, and suffered higher mortality than those reared on leaves dusted with non- 
transformed maize or on leaves without pollen. The conclusions of Losey et al. (1999) were 
challenged by other scientists on three grounds. First, the pollen doses used by Losey et al. 
(1999) were not quantitatively measured but were gauged by eye to match pollen dustings 
on milkweed leaves collected in the field. This raised concerns about subconscious biases on 
the part of the researchers. Secondly, concerns were raised as to the validity of extrapolating 
from the results of Losey at al. (1999), which considered only one type of pollen, to all types 
of Bt maize pollen. Lastly, the soundness of extrapolating from laboratory assays to the field 
was uncertain, although a subsequent field study by Jesse & Obrycki (2000) did seem to 
confirm the fears raised by the Losey et al. (1999) study (Minorsky, 2001). However, it 
should be emphasised that the continuous expression of the Bt toxins in the plant tissues 
throughout the growing period (Baumgarte & Tebbe, 2005) increases the chances and degree 
of exposure to non-target insects of ecological and economic importance. Hence, there are 
concerns about the possible adverse impacts of this novel pest control technology on the 
higher trophic level non-target arthropods (such as pollinators, pollen feeders and natural 
enemies of pests) through crop plant-based food chains. 

2.1 Bacillus thuringiensis and genetic engineering technology 

Bacillus thuringiensis is a soil-dwelling bacterium that produces large amounts of insecticidal 
5-endotoxin when it sporulates into a resting stage. This bacterium, abundantly found in 
grain dust from silos and other grain storage facilities, was discovered in Japan in 1901 by 
Ishawata (Baum et al., 1999). Bt is related to two other important spore-forming bacilli, B. 
cereus and B. anthracis and is differentiated largely on the basis of containing several 
plasmid-encoded protoxin genes (Aronson & Shai, 2001). There are hundreds of Bt 



324 Pesticides in the Modern World - Risks and Benefits 

subspecies and most produce, primarily during sporulation, one or more parasporal 
inclusions each comprised of either one or several related insecticidal protoxins, the so- 
called 5-endotoxins (Schnepf et al., 1998). These endotoxins are biologically inactive protein 
toxins that crystallize into characteristic shapes. In bacteria, the endotoxins are mixtures of 
several specific crystalline protein toxins (hence referred to as Cry proteins, Ostlie et al., 
1997) that are divided into several numbered classes; these are in turn subdivided into 
subclasses (Andow & Hilbeck, 2004). The mode of action of B. thuringiensis toxins (each of 
which is active on a subset of insect larvae from at least three orders of insects - Coleoptera, 
Lepidoptera, and Diptera, Gould and Keeton, 1996) involves ingestion followed by crystal 
solubilisation and proteolytic activation of protoxin in the insect midgut. Activated toxin 
binds to receptors in the midgut epithelial membrane and inserts into the membrane, 
leading to cell lysis and death of the insect (Schnepf et al., 1998). Because of unique but 
overlapping specificity profiles, Bt subspecies are generally effective against a broad range 
of insects, usually within a particular order of insects (Aronson & Shai, 2001). Also, many 
produce, during growth, less well characterized insecticidal proteins, the so-called 
vegetative insecticidal proteins (Estruch et al., 1996) as well as other pathogenic factors 
(Agaisee et al., 1999). 

Researchers have isolated the 5-endotoxin gene from different strains of Bt, and have 
expressed it in several crops in order to control lepidopteran and coleopteran pests (Groot & 
Dicke, 2002). Several of the isolated proteins have selective insecticidal properties against 
specific insect species (Andow & Hutchison, 1998). Therefore not all commercial Bt maize 
hybrids express the same insecticidal protein. Moreover, Bt maize plants may not express 
the protein uniformly throughout the plant, nor continuously throughout the crop season. 
Bt maize hybrids containing and expressing one of four proteins CrylAb, CrylAc or Cry9C, 
and CrylF have been developed and made available since 1996. Cry genes from B. 
thuringiensis are randomly inserted into plant chromosomes at different insertion sites via 
microprojectile bombardment using a particle gun technique (Bohorova et al., 1999). A 
promoter, a DNA sequence that regulates where, when, and to what degree an associated 
Cry gene is expressed (Ostlie et al, 1997), is attached to a Cry gene before it is inserted into a 
maize chromosome. A successful insertion of the new genetic package containing the 
modified Bt gene into a plant is called a transformation event (Rice & Pilcher, 1998). 
Different transformation events (in maize) provide varying levels of resistance to insect pest 
targets (Williams et al., 1997). 

2.2 Plant-Insect tritrophic systems and Bt crops 

Natural enemies have an important role to play in the co-evolution of plants and insects 
(Romeis et al., 2008a). "The third trophic level must be considered part of a plant's battery of 
defences against herbivores" (Price et al., 1980 as cited in Romeis et al., 2008a). Plant 
protection by natural enemies is well documented and has been manipulated in the 
development of biological control strategies in many crops (Dicke & Sabelis, 1988; Whitman, 
1994). Plants are well placed to influence the efficiency of parasitism and predation and they 
mediate numerous interactions between entomophagous arthropods and herbivores. Their 
structures and products often supply essential resources for parasitoids and predators. In 
addition, chemical and morphological plant attributes may affect the efficacy of biological 
control agents by influencing their abundance, survival, development time, fecundity and 



Transgenic Pesticidal Crops and the Environment: The Case of Bt Maize and Natural Enemies 325 

rate of attack (De Moraes et al., 2000). Moreover, plants influence the quality of parasitoids' 
herbivorous hosts by determining the quality of the host's nutrient intake (Vinson & 
Barbosa, 1987). Several studies show that secondary compounds ingested by the host affect 
parasitoids, either negatively or positively (De Moraes et al., 2000). Toxins and low 
nutritional quality may weaken the herbivore's immune system thus affecting its capacity to 
defend itself against parasitoid eggs (Benrey & Denno, 1997; Van den Berg & Van Wyk, 
2007; Vinson & Barbosa, 1987). 

In order to exploit arthropod herbivores, natural enemies must be able to locate small, 
highly dispersed targets within a complex spatial and chemical environment (De Moraes 
et al., 2000). Besides, herbivores have evolved numerous adaptations to avoid being 
discovered and attacked (Vet & Dicke, 1992). Plants provide both olfactory and visual 
signals used as foraging cues by parasitic and predaceous arthropods (Ma et al., 1992; 
Powell & Wright, 1991; Turlings et al., 1995). Some parasitoids use volatiles emitted by 
undamaged plants to locate the habitat and possibly microhabitat of their host (Ma et al., 
1992; Ngi-Song et al., 1996). Plant volatiles released in response to mechanical damage by 
herbivores are known to be attractive to various parasitoids (Mattiaci et al., 1994; 
Steinberg et al., 1993). Volatiles released in response to herbivore feeding are generally 
reliable indicators of herbivore presence and can potentially bring parasitoids in close 
proximity to their hosts (De Moraes et al., 2000). Indeed, plants are actively involved in 
the production and release of chemical cues that guide foraging parasitoids (Turlings et 
al., 1995). Therefore Bt maize may affect, negatively or otherwise, host finding through the 
volatile emissions. 

Extensive research has been published on the impacts of Bt plants on natural enemies within 
the context of agro-ecosystems (O 1 Callaghan et al., 2005; Romeis et al., 2006). Long-term, 
large scale field studies have indicated no meaningful impacts of Bt maize on predator 
populations even when the predator has acquired the toxin by feeding on intoxicated prey 
(Hellmich et al., 2005, as cited in Shelton et al., 2008). In addition, studies in which Bt crops 
were compared to conventional crops treated with insecticides have demonstrated the latter 
to be far more harmful to predators (Shelton et al., 2008). The situation, however, appears to 
be more complex for parasitoids. While an insect predator is characterised by feeding on 
multiple and various hosts during its lifetime, a parasitoid usually completes its entire 
lifetime within a single host and derives all its nutritional requirements by feeding on the 
host tissues. This intimate relationship between a parasitoid and its host would put the 
parasitoid at greater risk to any hazard its host encounters (Shelton et al., 2008). Parasitoids 
inside dead lepidopteran larvae that are exposed to B. thuringiensis usually suffer the same 
fate as the larvae. Thus, death of herbivore larvae caused by B. thuringiensis toxins may be 
detrimental to populations of parasitoids. Indeed, studies have found that herbivore larvae 
that were exposed to B. thuringiensis, but were themselves resistant to its effects, supported 
the normal development of parasitoids (Chilcutt & Tabashnik, 1999; Schuler et al., 1999a). 
Because the strains of B. thuringiensis currently in use are largely specific to Lepidoptera, 
there may be no direct consequences of B. thuringiensis on predators and parasites of 
herbivores (Agrawal, 2000). However, B. thuringiensis may have indirect negative effects on 
the populations of natural enemies of herbivores through the consumption of sick, dead, or 
dying herbivores (Agrawal, 2000). Critical questions that need to be considered in assessing 
the effect of Bt on natural enemies include: Do predators and parasitoids of herbivores avoid 
Bt exposed prey? Could behavioural mechanisms in parasitoids potentially reduce the 



326 Pesticides in the Modern World - Risks and Benefits 

indirect negative effect of Bt? Because the feeding of susceptible caterpillars on Bt plants is 
severely reduced, and plant damage attracts parasitoids, parasitoids may preferentially be 
attracted to either resistant larvae or susceptible larvae on Bt plants (Schuler et al., 1999b). 
Thus, a potential tri-trophic benefit of employing B. thuringiensis in agriculture is that 
parasitoids may act as agents for minimizing the evolution of resistance to B. thuringiensis in 
pests (Agrawal, 2000). 

Bt toxins may have indirect effects on beneficial insects such as parasitoids either by killing 
the intoxicated host (Schuler et al., 1999a), or rendering the host nutritionally unsuitable 
(Down et al., 2000). In turn, parasitoids' host quality may be influenced by host plants, 
giving rise to tri-trophic interactions (Price et al, 1980). For example, Ashouri et al. (2001) 
reported reduced weight of adult Aphidius nigripes Ashmead (Hymenoptera: Braconidae) 
developing on Macrosiphum euphorbiae Thomas (Homoptera: Aphididae) that was feeding on 
Bt potato. Other studies (e.g. Atwood et al., 1997a,b; Liu et al., 2004) showed that when host 
larvae were fed on a diet containing Bt protein, larval duration, pupal weight, body weight 
of the newly emerged adult, parasitoid emergence rates and adult longevity were negatively 
affected. Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae) developing inside 
Pseudoplusia includens Walker (Lepidoptera: Noctuidae) larvae that was feeding on Bt cotton 
suffered reduced longevity, and females had fewer ova (Baur and Boethel, 2003). Cotesia 
flavipes larval emergence was lower in Bt fed C. partellus larvae (23%), compared with non-Bt 
fed C. partellus, (83%) (Priitz & Dettner, 2004). Cocoon numbers and cocoon weight of 
parasitoids were decreased when Helicoverpa armirgera Hubner (Lepidoptera: Noctuidae) 
larvae fed on diet containing transgenic cotton leaf powder containing CrylA plus CpT (Ren 
et al., 2004). Liu et al. (2005a), in studies on the effects of transgenic cotton on Campoketis 
chloridae Uchida (Hymenoptera: Ichneumonidae) observed that the body weight of larvae of 
the parasitoids were significantly reduced when parasitized hosts fed on transgenic cotton 
leaves compared to those fed on traditional cotton. Duration of egg and larvae stage were 
significantly prolonged while pupal and adult weight of C. chloridae was decreased when 
the host larvae fed on transgenic cotton leaves longer than 48 hours. Development of the 
larval parasitoid, Microplitis mediator Haliday (Hymenoptera: Braconidae) was negatively 
affected when the host, H. armirgera, larvae were reared on a diet containing CrylAc toxin 
(Liu et al., 2005b). 

Nonetheless, a number of investigations show that Bt toxins are not pathogenic to 
parasitoids developing in infected hosts. For example, Orr & Landis (1997) observed that 
parasitism of European corn borer larvae by Eriborus terebrans Gravenhorst and Macrocentrus 
grandii Goidanich (Hymenoptera: Braconidae) was not significantly different in transgenic 
and non-transgenic plots. Schuler et al. (2004) observed that Cotesia plutellae Kurdjumov 
(Hymenoptera: Braconidae) eggs laid in Bt resistant Plutella xylostella Linnaeus (Lepidoptera: 
Plutellidae), fed on Bt oilseed rape leaves, developed to maturity and there was no effect of 
Bt plants on percentage parasitism, time to emergence from hosts, time to adult emergence, 
and percentage adult emergence from cocoons. Parasitoids reared on Bt susceptible hosts 
hatched, although premature host mortality did not allow the C. plutellae larvae to complete 
their development. This may support the thesis that the Bt toxin has no direct impacts on 
parasitoids, but rather that the impacts may be due to reduced host quality. Data so far 
indicate that parasitoids, in general, may be more susceptible to host quality and host- 
mediated impacts of GM crops compared with to predators (Lovei & Arpaia, 2005). 



Transgenic Pesticidal Crops and the Environment: The Case of Bt Maize and Natural Enemies 327 

From the foregoing, it is apparent that Bt plants may affect natural enemies either directly 
or indirectly. For the insecticidal proteins of insect resistant GM plants to directly affect an 
individual natural enemy, the organism has to not only be exposed to the toxin but also be 
susceptible to it. Thus, an organism is not affected by the GM plant when either exposure 
or sensitivity (hazard) does not occur. However, for an effect to be of ecological relevance 
it must result in changes in population or community processes. Similarly, direct or 
indirect effects of the GM plant on individual natural enemies, natural enemy species or 
groups/ guilds of natural enemies might not lead to a decreased biological control 
function (Romeis et al., 2008a). Moreover, natural enemies may be affected indirectly by 
the GM plant when they feed on sublethally impaired herbivores (sick prey). Such effects 
appear to be caused by declines in nutritional quality of the host/prey organism. These 
prey/host quality mediated effects appear to account for most (if not all) of the Bt plants' 
effects on natural enemies that have been reported from laboratory and glasshouse 
studies (Romeis et al., 2006). It is well established that parasitoids are especially 
vulnerable to changes in their hosts' quality, since they usually complete their 
development in a single host (Godfray, 1994). Therefore this review lays particular 
emphasis on the potential impacts of Bt maize on stem borer parasitoids. Bt maize are 
deployed to control Lepidoptera, which implies that lepidopteran parasitoid hosts would 
(as a direct consequence of being affected by the Bt toxin) invariably be less suitable for 
parasitoid development. Thus it is not surprising that parasitoid life-table parameters are 
significantly affected when the host suffers (Romeis et al., 2006). In extreme cases, 
parasitoids attack sublethally affected hosts that die before the parasitoid offspring 
completes development (Davidson et al., 2006; Schuler et al., 2004). Sections 2.2.1 to 2.2.5 
provides a review of the potential impacts of Bt maize on stem borers and their natural 
enemies (specifically parasitoids). 

2.2.1 Effect of Bt maize on stem borer oviposition preference 

One of the major risks associated with the use of transgenic pesticidal crops is that pests can 
develop resistance which could reduce the efficacy of such crops as plant protection tools 
(Wolfenbarger & Phifer, 2000). Furthermore, if larvae developed resistance to the Bt toxin, 
there could be greater chances of natural enemies getting host-mediated exposure to the 
toxin (Obonyo et al., 2008a). When the US EPA reviewed the first registration for Bt plants, 
there was considerable concern in some sectors that resistance to the plants would rapidly 
occur and that not only would this be a concern to growers of Bt crops but also to organic 
farmers who relied on Bt as a foliar spray (Shelton et al, 2008). The high dose/ refuge 
strategy (the use of high doses of one or more toxins, combined with a refuge of non-Bt 
plants) has been proposed as a likely means to delay the development of resistance by 
insects against transgenic plants (Bates et al., 2005). This strategy emphasises the presence of 
susceptible insect populations; these may slow down the evolution of resistance (Bentur et 
al, 2000; Shelton et al., 2000; Tang et al., 2001). The premise is that susceptible insects, if 
present in sufficient numbers, would mate with resistant insects and dilute resistance genes. 
However, several biological factors that influence the number of insects exposed to Bt toxin 
may substantially affect the success of the high dose/refuge strategy (Ives & Andow, 2002). 
One such factor is oviposition preference. Preference for Bt maize would require more 
refuge plants to counter an increased selection pressure. However, preference for refuge 
plants could have the opposite effect. From a resistance management perspective, an ideal 



328 Pesticides in the Modern World - Risks and Benefits 

plant, in addition to killing larvae, should repel adult oviposition (Hellmich et al., 1999). 
This would reduce selection for resistance because fewer larvae would be exposed to plant 
toxins. Potential effects of Bt transgenic maize on stem borer natural enemies could therefore 
partly depend on the oviposition preferences of stem borers, either for Bt or non-Bt maize. 
Various studies have been conducted on effects of Bt maize on stem borer oviposition 
behaviour. In field tests, the number of eggs laid by susceptible European corn borer 
females did not differ between Bt corn (containing CtylAb) and non-Bt corn (Orr & Landis, 
1997). Pilcher & Rice (2001) observed that O. nubilalis females did not show any oviposition 
preference towards non-Bt or Bt maize (using Event 176 and Btll). Van den Berg & van Wyk 
(2007) reported that S. calamistis adults did not differentiate between Bt and non-Bt maize 
plants in oviposition choice experiments. More recently, Obonyo et al. (2008a) observed that 
C. partellus and S. calamistis moths did not discriminate between Bt and non-Bt maize plants 
for egg laying. This non-discriminatory oviposition behaviour could be due to the fact that 
the ratios of caterpillar-induced odour emissions of Bt maize plants are identical to those of 
non-Bt plants (Turlings et al, 2005) since genetic modification does not alter the volatile 
profile of undamaged maize plants (Dean & De Moraes, 2006). These results have important 
implications for pest resistance management and monitoring. Because oviposition is not 
affected by the Bt toxin, and females are exposed equally to Bt maize and non-Bt maize 
refuges, it can be assumed that eggs will be distributed equally between Bt and non-Bt 
maize hence there will always be a pool of insects on susceptible crops, which is necessary 
for resistance management and hence ensuring that the development of resistance is 
delayed as much as possible. Furthermore, since the development of resistance against Bt 
toxins requires the survival and development of at least two exposed larvae into a male and 
a female (Kumar, 2004) and since Bt maize causes up to 100% mortality (Obonyo et al., 
2008a) the possibility of resistance development would be further restricted. 

2.2.2 Effect of Bt maize on stem borer development and mortality 

An understanding of the effect of Bt toxins on development of herbivorous insects is 
important because host development time could have a direct effect on natural enemies by 
influencing the 'window of vulnerability', the period during which the host is exposed to 
natural enemies (Schoenmaker et al., 2001; Schuler et al., 1999a; Wallner et al,. 1983). Also, 
the combined effects of developmental delays may result in temporal asynchrony of moths 
emerging from Bt and non-Bt maize- resulting in susceptible individuals mating before 
resistant adults emerge (Horner et al., 2003). Since the success of the refuge strategy requires 
that any resistant individuals mate with susceptible ones, such asynchrony in emergence 
from Bt and non-Bt maize plants could compromise the strategy and hence weaken the 
potential of Bt maize as an option for stem borer control. Furthermore, effects of Bt plants on 
host development could impact on the biology of a natural enemy developing in such a host 
(Walker et al, 2007; Weseloh, 1984). 

Obonyo et al. (2008b) observed that Bt maize had significant effects on stem borer 
development time. Feeding of stem borer larvae on Bt plant tissue at the 3 rd and 4 th instars 
significantly lengthened the duration of the respective instars (but not the subsequent 
ones) while overall larval development time was not affected probably because the larvae 
were exposed to Bt for a relatively short duration. Schoenmaker et al. (2001) suggested 
that ingestion (by lepidopteran larvae) of sublethal doses of Bt toxin prolonged 
development time by temporarily inhibiting feeding. Continuous exposure to Bt toxin 



Transgenic Pesticidal Crops and the Environment: The Case of Bt Maize and Natural Enemies 329 

prolonged development of Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) while 
exposure to toxin for shortened durations had no significant effects on larval development 
time (Dutton et al., 2005a). Therefore it seems that larvae may recover from the effects of 
the Bt-toxin, following transient exposure. Other lepidopteran larvae that ingest sublethal 
doses of Bt also resume normal development after a few days (Moreau and Bauce, 2003; 
Siegfried et al., 2001). Dutton et al. (2005a) reported that there were no significant effects 
on overall larval development when 3 rd instars of S. littoralis larvae were exposed to Bt 
sprayed plants because the effect of the toxin did not persist for long due to rapid 
degradation of the Bt spray (Haddad et al v 2005). In contrast, significant effects, attributed 
to long toxin persistence, were reported when larvae were reared for four days on Bt 
maize (Haddad et al., 2005). Huang et al. (2006) observed larval development inhibition of 
O. nubilalis, D. grandiosella and Diatraea saccharalis F. (Lepidoptera: Pyralidae) fed on a diet 
prepared from CrylAb protein (extracted from Bt corn leaves). Similarly, transgenic maize 
containing CrylAb delayed larval development of H. zeae (Horner et al., 2003; Stewart et 
al., 2001) and D. plexippus (Dively et al., 2004). Development time of the 5 th instar of C. 
partellus larvae subjected to transient feeding on Bt maize at the same growth stage was 
not affected (Obonyo et al., 2008b), possibly because pupation follows shortly after the 5 th 
larval stage in this species at which time the larvae are relatively inactive and do not feed 
much (Tettamanti et al., 2007); and their large sizes enable them to tolerate more toxin 
(Huang et al., 1999). Overall, larval development time in these larvae was significantly 
longer as a consequence of Bt exposure (Obonyo et al., 2008b). This indicates a 
disturbance to the "normal" development cycle, from which the larvae may eventually 
recover. The increase in larval development time therefore increases the window of 
vulnerability during which stem borer parasitoids can get host mediated exposure to the 
Bt toxin. This in itself may not be cause for concern but rather the consequences of such 
exposure. Possible consequences of host-mediated exposure to Bt toxins are discussed in 
subsequent sections of this chapter. 

2.2.3 Effect of Bt maize on the ability of parasitoids to locate hosts 

The success of biological control agents depends on their efficiency to search for, and locate 
target hosts (Nordlund et al., 1988). Parasitoid host finding behaviour is complex and 
influenced by many factors (Ngi-Song et al., 1996; van Leerdam et al., 1985). One important 
factor is volatiles emitted by the host plant. There are significant quantitative (Turlings et al., 
2005) and qualitative (Dean and De Moraes, 2006) differences in volatile emissions between 
Bt and non-Bt plants. Both the quantity and composition of emitted volatiles influence host 
finding by Cotesia species (Steinberg et al., 1993). Host species odours are also used by 
parasitoids for host location and hence any change in host physiology may alter parasitoids' 
host location behaviour (Takasu & Lewis, 2003). 

Bt maize may influence host species odours and thus parasitoid host finding behaviour. 
Obonyo (2009) showed that damaged but uninfested Bt and non-Bt maize were similarly 
attractive to females of the larval parasitoids C. flavipes and C. sesamiae, and that both were 
also more attractive than the control air flow from a plantless cage. Females of C. flavipes and 
C. sesamiae were equally attracted to stem borer infested maize plants (irrespective of Bt 
status). This suggests that females of C. flavipes and C. sesamiae do not distinguish among 
plant- and host-derived cues from Bt and non-Bt maize when searching for stem borer hosts. 
Therefore the presence of Bt toxin in maize plants apparently did not affect the host location 



330 Pesticides in the Modern World - Risks and Benefits 

process of these parasitoids. Similar findings have been reported elsewhere (Ngi-Song and 
Overholt, 1997; Potting et al., 1997). Also, Cotesia marginiventris Cresson (Hymenoptera: 
Braconidae) and Microplitis rufiventris Kokujev (Hymenoptera: Braconidae), which are 
important larval parasitoids, were not able to distinguish between the odours of a Bt maize 
event and its near-isogenic line (Turlings et al., 2005). This indicates that growing Bt maize is 
not likely to affect host finding by stem borer larval parasitoids. 

Furthermore, plant volatiles may act as cues for host location by pupal parasitoids (Obonyo 
2009). Chemical analyses of collected odours between Bt and non-Bt maize revealed 
significant quantitative differences (Turlings et al., 2005); this could possibly affect host 
location by pupal parasitoids such as X. stemmator. Indeed, Obonyo (2009) found that X. 
stemmator parasitoids preferred host plant odours compared to odours from a blank control. 
However, volatiles from Bt plants were deterrent to X stemmator. Oviposition preference of 
insects has been predicted to correlate with host suitability for offspring development 
(preference - performance hypothesis) (Jaenike, 1978). This hypothesis (known as the 
'mother knows best 1 principle) (Johnson et al., 2006) was developed for herbivorous insects 
but is assumed to play an important role in parasitic Hymenoptera as well (Vinson and 
Iwantsch, 1980). According to this preference - performance hypothesis, X. stemmator avoids 
the Bt plants because potential hosts have a lower quality when feeding on Bt maize. In 
parasitoids, host organisms are the only source of nutrients for the immature stages 
(Sequeria & Mackauer, 1992), and thus parental fitness depends on the accurate assessment 
of host sites for their potential to sustain the development of their larvae (Meyling & Pell, 
2006). Therefore adaptation to reliable cues, enabling the evaluation of the quality of 
potential hosts, is a selective advantage for ovipositing females. Chemical cues may not only 
attract but also deter parasitoids from entering host sites. Naive females of the solitary 
ectoparasitoid, Lariophagus distinguendus Forster (Hymenoptera: Pteromalidae) which 
parasitizes immature stages of several stored-product infesting beetle species avoid odours 
from mouldy grains which are unsuitable for the development of their larvae (Steiner et al., 
2007). 

Considering that Bt maize has an adverse effect on the host location behaviour of X 
stemmator, it could possibly compromise the biocontrol potential of this parasitoid, hence 
impacting negatively on the use of Bt maize as part of IPM strategies. 

2.2.4 Effect of Bt maize on parasitoid biology 

Changes in host plant chemistry may negatively affect natural enemy fitness through 
reducing survivorship, clutch size, body size and/ or fecundity (Ode, 2006). Such negative 
impacts may occur either directly (when the natural enemy encounters the toxin in its host 
or prey) or indirectly (when natural enemy fitness is reduced due to lower prey/ host size or 
quality). A number of studies have found variable results on effects of Bt toxins on 
parasitoid life history parameters; these include no apparent negative effect (Obonyo, 2009; 
Schuler et al., 1999a), synergism between the transgenic plants and parasitoids (Tounou et 
al, 2007), lower parasitoid survival (Blumberg et al., 1997), or emergence rates (Atwood et 
al, 1997b; Liu et al., 2005a), increased parasitoid development times (Liu et al., 2005a; Liu et 
al, 2005b; Vojtech et al., 2005), reduced longevity (Baur & Boethel, 2003), reduced body mass 
(Ashouri et al., 2001; Liu et al., 2005a), and altered parasitoid sex ratios (Wallner et al., 1983). 
Changes in host plant chemistry may also affect acceptance of the plants by their hosts, with 
consequences on associated natural enemies. Obonyo (2009), however, showed that there were 



Transgenic Pesticidal Crops and the Environment: The Case of Bt Maize and Natural Enemies 331 

no significant differences in host acceptance ratio between Bt exposed and non-Bt reared 
larvae. Turlings et al. (2005) observed that braconid parasitoids did not distinguish between 
odours of Bt and non-Bt maize plants in olfactometer experiments. Although a number of 
studies have found no significant differences in oviposition choice between hosts fed on 
transgenic and non-transgenic diets (Bell et al., 1999, Schoenmaker et al., 2001; Schuler et al., 
1999b), there are cases where parasitoids seem to distinguish by host quality (reviewed in 
Steidle and van Loon, 2003; Overholt et al., 1994; Sallam et al, 1999). More recently, Obonyo 
(2009) reported a higher host acceptance ratio of C. flavipes for C. partellus compared with S. 
calamistis. These contrasting results could be due to the different dietary material (plant 
material and microbial formulations) used in the various studies. However, it is more likely 
that the lack of significant effects of Bt was due to the transient feeding of the host on Bt maize 
(Obonyo, 2009). Negative effects of Bt toxins on parasitoids are often indirect, occurring via 
reduced host quality (Chen et al, 2008; Vojtech et al, 2005; Walker et al, 2007) but larvae 
exposed to the Bt toxin and subsequently transferred to a Bt-free diet may recover by replacing 
damaged mid-gut cells and excreting the toxin (Tounou et al., 2007). 

As already mentioned, host quality directly impacts on parasitoid development. Ingestion 
of Bt toxins by stem borer larvae could therefore affect parasitoid developing within these 
larvae. Temerak (1980), Salama et al. (1991) and Atwood et al. (1997b) observed that 
incorporation of microbial Bt formulations in host food decreased the emergence of 
parasitoids larvae. Wanyama (2004) found that Bt contaminated diets significantly 
increased C. partellus cocoon development time. Bernal et al. (2002) found a longer 
development time of Parallorghas pyralophagus Marsh (Hymenoptera: Braconidae) on Bt 
fed hosts. In contrast, Obonyo (2009) and Prutz & Dettner (2004) observed no effects of 
host ingested Bt toxins on the mortality of C. flavipes inside cocoons. Also, Prutz & Dettner 
(2004) found no significant effects of Bt-contaminated diets on C. flavipes pre-cocoon 
development time. 

Besides, the proportion of female parasitoids produced in each generation is an important 
factor in the success and survival of parasitoid populations (Godfray, 1994). A female 
biased sex ratio is an important characteristic of a biocontrol age